Targeting the human immunodeficiency virus type-1

Gag protein into the defective ribosomal product

pathway enhances its MHC class I

antigen presentation

Die Bedeutung fehlerhafter ribosomaler Produkte

für die MHC Klasse I Antigenpräsentation

des humanen Immundefizienzvirus-1

Strukturproteins Gag

Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität

Erlangen-Nürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Sabine Hahn aus Regensburg

Als Dissertation genehmigt

von der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: ..................... 25.07.2011..........…………..

Vorsitzender der Promotionskommission: .... Prof. Dr. Rainer Fink…........

Erstberichterstatter: ...................................... Prof. Dr. Ulrich Schubert…..

Zweitberichterstatter: .................................... Prof. Dr. Robert Slany……..

Table of contents

1 Abstract .......................................................................................................................5

2 Zusammenfassung.......................................................................................................6

3 List of abbreviations....................................................................................................7

4 Introduction ...............................................................................................................10

4.1 The human immunodeficiency virus type 1......................................................10

4.2 Gag proteins and their role in late processes of HIV-1 replication...................12

4.3 The ubiquitin proteasome system .....................................................................13

4.4 Role of the UPS in late steps of HIV-1 replication...........................................17

4.5 Role of the UPS and defective ribosomal products (DRiPs) in MHC-I antigen

processing ......................................................................................................................20

4.6 Regulation of UPS-mediated proteolysis by degradation signals .....................22

4.7 Correlation between metabolic half-life and MHC-I antigen presentation.......24

5 Results .......................................................................................................................25

5.1 Targeting HIV-1 Gag into the DRiP-pathway enhances MHC-I antigen

presentation and CD8+ T-cell activation........................................................................25

5.1.1 Construction of Gag variants containing degradation signals ......................25

5.1.2 Introduction of the OVA-derived SL epitope as indicator for Ag processing

of Gag ......................................................................................................................26

5.1.3 Generation and characterization of GagSL-expressing EL4 cell lines .........27

5.1.4 Half-life and DRiP-rate of UbRGagSL and UbMGagSL proteins ...............28

5.1.5 Correlation of DRiP-rate with the MHC-I presentation of Gag-derived SL.31

5.1.6 In vitro activation of the SL-H2-Kb specific T-cell hybridoma B3Z............33

5.1.7 In vivo activation of SL-H2-Kb-specific OT-1 cells and induction of SL-

specific CD8+ T cells in naïve mice ..........................................................................35

5.1.8 In human cells, Gag is targeted into the MHC-I pathway by the N-end rule,

but even more efficiently by stable N-terminal fusion to Ub....................................37

5.1.9 N-end rule and UFD degradation signals do not influence the synthesis or

metabolic half-life of Gag in HeLa cells ...................................................................39

5.1.10 N-end rule and UFD degradation signals interfere with the release of

VLPs ..................................................................................................................41

5.1.11 N-end rule and UFD degradation signals disturb the membrane

localization of Gag ....................................................................................................43

5.2 The PTAP Late domain regulates ubiquitination and MHC-I antigen

presentation of HIV-1 Gag ............................................................................................46

5.2.1 The PTAP L-domain in the p6 region regulates budding of GagSL-derived

VLPs. ......................................................................................................................46

5.2.2 The PTAP L-domain regulates ubiquitination of GagSL .............................47

5.2.3 The PTAP, but not the YP(X)nL L-domain regulates MHC-I antigen

presentation of a Gag-derived epitope.......................................................................48

5.2.4 Induction of the immunoproteasome enhances presentation of the SL-epitope

derived from GagSL-GFP .........................................................................................51

5.2.5 The PTAP L-domain regulates MHC-I antigen presentation of the SL

epitope derived from processed Gag .........................................................................52

5.2.6 Enhanced SL-presentation of the PTAP-mutant is not a result of the budding

defect and not entirely dependent on membrane association of Gag ........................55

5.2.7 The interaction with Tsg101 or ALIX is not essential for the regulation of

MHC-I presentation of a Gag-derived epitope by the PTAP L-domain ...................56

5.2.8 Lys48-linked polyubiquitination is essential for the preferred entry of the

PTAP-mutant into the MHC-I pathway ....................................................................58

5.2.9 The PTAP-mutant displays a slightly decreased metabolic half-life and an

increased DRiP-rate when compared to wt Gag........................................................59

6 Discussion .................................................................................................................62

7 Material and methods ................................................................................................73

8 References .................................................................................................................81

9 Acknowledgements ...................................................................................................98

Abstract 5

1 Abstract

The major source for endogenous peptides presented via the major histocompatibility

complex class-I (MHC-I) pathway are de novo synthesized, dysfunctional proteins,

named defective ribosomal products (DRiPs), which are degraded in concert with or

shortly after their synthesis by the ubiquitin proteasome system (UPS).

The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, a bona fide

substrate of the DRiP-pathway, was chosen as a model antigen to more precisely

understand the relevance of erroneous protein synthesis for the generation of MHC-I-

presented peptides. To target Gag into the DRiP-pathway, various degradation signals

have been introduced into Gag, and their effects on its protein synthesis, metabolic half-

life, DRiP-formation as well as subcellular localization and the release of virus like

particles have been investigated. As an indicator for antigen processing, the ovalbumin-

derived SIINFEKL (SL) epitope was introduced into Gag expressed from a codon-

optimized gag gene (syngag). It was demonstrated that exchange of the N-terminal Met

residue for Arg (RGag), a destabilizing amino acid according to the N-end rule, directed

Gag more efficiently into the DRiP-pathway in murine EL4 cell lines. This correlated

with enhanced MHC-I antigen presentation as well as more efficient CD8+ T-cell

activation in vitro and in vivo. The enhanced MHC-I presentation of SL derived from

RGag in murine cells could be reproduced in a human cell line. Furthermore, stable

fusion to ubiquitin (Ub), converting Gag into a substrate for the Ub fusion degradation

(UFD) pathway, was even more efficient in targeting Gag into the MHC-I pathway.

The PTAP late (L)-domain motif in the p6 domain of HIV-1 Gag plays an essential role

during late stages of budding and has been recently implicated in the control of Gag

ubiquitination. Mutations of PTAP in the context of syngag- or HIV-1-encoded Gag

increased the ubiquitination as well as the DRiP-rate of Gag and enhanced the MHC-I

presentation of the Gag-derived SL epitope. This novel function of the PTAP L-domain

as a naturally occurring motif that regulates the DRiP-rate of Gag might be mediated by

the sequence-specific recruitment of cellular factors, most likely components of the UPS.

Altogether, the results presented in this study further underline the role of the DRiP-

pathway in adaptive immunity and provide strategies to enhance the MHC-I antigen

presentation of HIV-1 Gag and other antigens. It remains to be elucidated by studies

performed in vivo whether such approaches may help to improve vaccination strategies.

Zusammenfassung 6

2 Zusammenfassung

Die Hauptquelle für endogene Peptide, die von MHC Klasse I (MHC-I) Molekülen

präsentiert werden sind Fehlprodukte der Proteinbiosynthese, sogenannte defekte

ribosomale Produkte (DRiPs), die noch während oder kurz nach ihrer Synthese durch das

Ubiquitin-Proteasom-System (UPS) abgebaut werden.

Um die Bedeutung der fehlerhaften Proteinsynthese für die MHC-I Antigenpräsentation

weiter zu untersuchen, wurde das Gag Polyprotein des humanen Immundefizienzvirus-1

(HIV-1), ein beschriebenes Substrat des DRiP-Pathways, als Modellantigen gewählt. Um

Gag in den DRiP-Pathway zu lenken, wurden verschiedene Abbausignale eingeführt und

deren Wirkung auf die Synthese, metabolische Halbwertszeit, DRiP-Rate sowie

subzelluläre Lokalisation von Gag und die Freisetzung von Virus-ähnlichen Partikeln

untersucht. Als Indikator für die Antigenprozessierung wurde das SIINFEKL (SL) Epitop

aus Ovalbumin in Gag eingebracht, welches von einem synthetischen, Kodon-optimierten

gag Gen (syngag) exprimiert wurde. Es wurde gezeigt, dass der Austausch des N-

terminalen Methionins durch Arginin (RGag), welches entsprechend der „N-end Regel“

ein Abbausignal darstellt, zu verstärkter Bildung von Gag-DRiPs in murinen EL4 Zellen

führt. Dies korrelierte mit erhöhter MHC-I Antigenpräsentation sowie einer effizienteren

T-Zellaktivierung in vitro und in vivo. Die bessere Antigenprozessierung von RGag in

murinen Zellen konnte in einer humanen Zellinie bestätigt werden. Darüber hinaus leitete

eine stabile N-terminale Fusion von Ubiquitin das Protein noch weitaus effizienter in den

MHC-I Pathway.

Das PTAP late (L)-Domänen Motiv in der p6 Domäne von HIV-1 Gag spielt eine

essentielle Rolle in späten Stadien der Virusfreisetzung und reguliert die

Ubiquitinylierung von Gag. Mutationen von PTAP im Kontext von syngag- oder HIV-1

kodiertem Gag erhöhen die Ubiquitinylierung und DRiP-Rate und steigern die MHC-I

Präsentation des SL-Epitops. Diese neu beschriebene Funktion des PTAP Motivs als ein

inhärentes Sequenzmotiv, welches den Eintritt von Gag in den MHC-I Pathway reguliert,

könnte durch die sequenzspezifische Interaktion mit zellulären Faktoren, insbesondere

Bestandteile des UPS, vermittelt werden. Zusammengefasst unterstreichen diese Befunde

die Rolle des DRiP-Pathways in der adaptiven Immunität and zeigen mögliche Strategien

auf, mit Hilfe derer die MHC-I Präsentation von HIV-1 Gag oder anderen Antigenen

gesteigert werden könnte. Dennoch bedarf es weiterer Untersuchungen in vivo, um zu

klären, ob auf diese Weise Vakzinierungsstrategien verbessert werden könnten.

List of abbreviations 7

3 List of abbreviations

Standard three letter abbreviations are used for amino acids.

aa amino acid(s) AAA ATPase ATPase associated with various cellular activities Ab antibody AIDS acquired immunodeficiency syndrome ALG2 Apoptosis-Linked Gene 2 ALIX ALG2 interacting protein X Ag antigen APC antigen presenting cell APC allophycocyanin Ate arginyl-tRNA-protein transferase β2m beta2-microglobulin BCA bicinchoninic acid β-Gal β-Galactosidase BSA bovine serum albumin CA capsid CCR CC motif chemokine receptor CCT chaperonin containing TCP-1 CD cluster of differentiation CFSE Carboxyfluoresceine succinimidyl ester CHMP charged MVB proteins CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-

propanesulfonate CMV cytomegalovirus CP core particle CRT Calreticulin CTL cytotoxic T lymphocyte CXCR C-X-C chemokine receptor type Da Dalton DC dendritic cell DIAP1 Drosophila inhibitor of apoptosis DMEM Dulbecco´s modified Eagle medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dpi day postinfection DUB deubiquitinating enzyme DTT dithiothreitol EBV Epstein-Barr virus ECL enhanced chemiluminescence EIAV equine infectious anemia virus ELISA Enzyme linked immunosorbent assay ELISPOT Enzyme linked immunospot technique Env Envelope ER endoplasmic reticulum ERAAP ER aminopeptidase associated with Ag processing ERAD endoplasmic reticulum-associated degradation ESCRT endosomal sorting complex required for transport

List of abbreviations 8

FCS fetal calf serum FIV feline immunodeficiency virus Gag group specific antigen HA hemagglutinin HAART highly active antiretroviral therapy HECT Homologous to E6-associated protein C-terminus HIV human immunodeficiency virus HRP horseradish peroxidase HTLV human T cell leukemia virus IAV influenza A virus IFN Interferon Int Integrase IP immunoprecipitation ISG Interferon stimulated gene JAMM Jab1/MPN metalloenzyme kbp kilo base pairs kDa kilo Dalton LC lactacystin LCMV lymphocytic choriomeningitis virus L-domain late domain LN lymph node LTR long terminal repeat MA Matrix MetAP Methionine aminopeptidase MFI mean fluorescence intensity MHC-I major histocompatibility complex class I MHR major homology region MJD Machado-Joseph domain MLV murine leukemia virus MMTV mouse mammary tumor virus MoMLV Moloney murine leukemia virus MPMV Mason-Pfizer monkey virus mRNA messenger RNA MVB multivesicular body MW molecular weight NA Neuraminidase NC Nucleocapsid NME N-terminal Met excision NO nitric oxide NP Nucleoprotein NTA N-terminal amidases ORF open reading frame OTU ovarian-tumor OVA ovalbumin PAGE polyacrylamide gel electrophoresis pAPC professional antigen presenting cell PBS phosphate buffered saline PCR polymerase chain reaction PE Phycoerythrin PFA paraformaldehyde PHA phytohemagglutinin PI proteasome inhibitor

List of abbreviations 9

PLC peptide loading complex PM plasma membrane pMHC peptide-MHC complex Pol Polymerase POSH plenty of SH3 PR Protease PSI protein biosynthesis inhibitor PVDF polyvinylidenefluoride RGS regulator of G-protein signaling RING Really interesting new gene RIPA Radioimmunoprecipitation assay RNA ribonucleic acid RNAi RNA interference RP regulatory particle RSV Rous sarcoma virus RT Reverse transcriptase rVV recombinant vaccinia virus SD standard deviation SDS sodium dodecyl sulfate SEA staphylococcal enterotoxin A siRNA small interfering RNA SIV simian immunodeficiency virus SL SIINFEKL SUMO small Ub-related modifier TAP transporter associated with antigen processing TCR T cell receptor TGN trans-Golgi network TH tyrosine hydroxylase TM transmembrane TOP Thimet oligopeptidase TPPII tripeptidyl peptidase II TRiC tailless complex polypeptide-1 (TCP-1) ring complex TRIM Tripartite interaction motif TRP-2 tyrosinase-related protein-2 Tsg101 tumor susceptibility gene 101 Ub ubiquitin UBL ubiquitin-like UCH ubiquitin-C-terminal hydrolase UEV ubiquitin enzyme 2 variant UFD ubiquitin fusion degradation UPS ubiquitin proteasome system USP ubiquitin-specific proteases VLPs virus like particles zLLL carbobenzoxyl-leucine-leucine-leucinal

Introduction 10

4 Introduction

4.1 The human immunodeficiency virus type 1

The human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired

immunodeficiency syndrome (AIDS), first described in 1981 (1). The number of people

living with an HIV-1 infection world-wide is still increasing and has reached 33.3 million

in 2009, with 2.6 million people that were newly infected with HIV-1 and 1.8 million

people dying from AIDS, as estimated by the World Health Organization of the United

Nations. Although the introduction of highly active antiretroviral therapy (HAART) in the

mid nineties has significantly reduced morbidity and mortality among AIDS patients,

eradication of the virus from infected individuals has not been achieved and the disease

remains incurable. With still very limited access to HIV-1 prevention and treatment in

developing countries, the HIV-1 pandemic remains one of the most critical of infectious

disease challenges to public health.

Fig. 4.1: Replication cycle of HIV-1 (artwork by Nadine Jänisch). Schematic representation of the major steps in HIV-1 replication. The replication cycle of HIV-1 begins with the attachment of the virus particle to CD4 and one of the coreceptors CXCR4 or CCR5, followed by membrane fusion, virus entry and uncoating. Following reverse transcription, the proviral DNA is integrated into the host cell genome. The late steps of replication start with the transcription of viral genes and the de novo synthesis of viral structural proteins which undergo assembly and budding at the plasma membrane. Following auto-catalytic activation, the viral protease processes the structural proteins resulting in the formation of a conical core that is typical for a mature, infectious virus particle.

Introduction 11

HIV-1 belongs to the lentivirus subfamily of the Retroviridae, a family of enveloped

RNA viruses, that, instead of using their RNA genome directly for virus replication,

reversely transcribe it into proviral DNA that is integrated into the host cell genome. The

retroviral life cycle, depicted in Fig. 4.1. begins with the binding of infectious viral

particles to cellular receptors, in case of HIV-1 CD4 and one of the coreceptors CCR5

(CC chemokine receptor 5) or CXCR4 (C-X-C chemokine receptor type 4), followed by

membrane fusion and virus entry. After reverse transcription of the RNA genome and

integration of the proviral DNA into the host chromosomes, virus proteins are synthesized

and assemble at the plasma membrane (PM). During budding, the Gag polyprotein

precursor is processed by the viral protease (PR) after its auto-catalytic activation,

resulting in the formation of a conical core which is typical for the mature and infectious

HIV-1 particle.

Fig. 4.2: Genomic organization of HIV-1. This figure depicts the complex organization of the HIV-1 genome comprising the canonical retroviral ORFs flanked by long terminal repeat (LTR) sequences: gag (blue) coding for the polyprotein precursor Pr55 that is processed into the individual structural Gag proteins, pol (green) encoding the enzymes Protease, Reverse Transcriptase, RNaseH and Integrase and env (orange), coding for the glycoprotein precursor gp160 that is processed into gp120 and gp41. Six additional genes code for regulatory proteins that are classified as essential (Tat and Rev) or accessory (Vpr, Vif, Vpu, Nef). A schematic view of a mature HIV-1 virus particle with the incorporated proteins and two copies of RNA is shown below. The HIV-1 genome is approximately 9 kbp in length and contains the three open reading

frames (ORFs): gag encoding the structural Gag (group specific antigen) proteins, pol

coding for the viral enzymes PR, Reverse transcriptase (RT), RNase H and Integrase (Int)

and env, which encodes the surface glycoproteins gp 41 and gp 120. In addition to those

canonical retroviral coding regions, HIV-1, as a complex retrovirus, encodes six

Introduction 12

regulatory proteins: Tat and Rev, which are essential for efficient transcription and the

nuclear export of single- or unspliced viral mRNA, respectively, and the accessory

proteins Vpr, Vpu, Vif and Nef, which are dispensable for viral replication in cell culture,

but play important roles during infection in vivo (2). A schematic overview of the HIV-1

genome and a virus particle is depicted in Fig. 4.2.

4.2 Gag proteins and their role in late processes of HIV-1

replication

The virus structure proteins Gag are synthesized from an unspliced RNA in the cytoplasm

as a polyprotein precursor, named Pr55 according to its molecular weight (MW). During

five to ten percent of all translation events, ribosomal frameshifting into the pol reading

frame occurs (3), resulting in the synthesis of a Pr160 Gag-Pol fusion protein. Pr55

undergoes cotranslational myristoylation at an N-terminal Gly residue, which is crucial

for binding to membranes and hence, for virus assembly and budding (4). During

maturation, which occurs concomitantly with or shortly after virus release, the Gag

precursor is cleaved by the viral PR into the individual structural proteins Matrix (MA,

p17), Capsid (CA, p24), Nucleocapsid (NC, p7), p6 and two spacer peptides p1 and p2.

However, Pr55 alone is both essential and sufficient for the assembly and release of virus

like particles (VLPs; (5)).

At least three conserved and interchangeable domains essential for virus assembly have

been identified in Pr55: the membrane binding domain (M), the interaction domain (I)

and the late domain (L). Membrane binding is mediated by the myristate group and basic

residues clustered in the N-terminal region of MA (6). The I-domain is required for the

assembly of particles of normal density and has been mapped to the NC domain (7). The

L-domain is essential for the final abscission of the budded virion from the PM. L-

domains have been identified in virtually all retroviral Gag proteins as well as other

enveloped viruses like filoviruses, rhabdoviruses and arenaviruses (8, 9). The fact that, in

some cases, L-domains can function independently from their position within Gag

proteins and can even be switched between different viruses (10), suggests that they serve

as docking sites for cellular factors to promote virus release, and several of those have

been identified.

The C-terminal p6 region of HIV-1 Gag contains two distinct L-domains. The primary L-

domain has been mapped to the tetrapeptide motif PTAP and promotes budding via its

interaction with the UEV (Ub enzyme 2 variant) domain of Tsg101 (tumor susceptibility

Introduction 13

gene 101), a component of the cellular endosomal sorting complex required for transport-

I (ESCRT-I; (11-13)). Prevention of this interaction abolishes HIV-1 release almost

completely (11, 12), as does overexpression of Tsg101 (14) or an N-terminal fragment

thereof (15). PT/SAP L-domain motifs are also found in other retroviruses like HIV-2,

simian immunodeficiency virus (SIV), human T cell leukemia virus (HTLV), Moloney

murine leukemia virus (MoMLV), Mason-Pfizer Monkey Virus (MPMV), feline

immunodeficiency virus (FIV) and Ebola virus, a filovirus (12, 16).

More C-terminally within HIV-1 p6, a degenerated version (YP(X)nL) of the YPDL L-

domain motif, which among others is also present in equine infectious anemia virus

(EIAV), binds to the central V-domain of the ESCRT-associated adaptor protein ALIX

(ALG (apoptosis linked gene)-2-interacting protein X; (17-20)). The release of PTAP L-

domain mutants harboring an intact ALIX binding site can be rescued by overexpression

of ALIX (18, 21, 22). While mutation of the ALIX binding site within p6 has only a mild

effect on wt HIV-1 particle release, overexpression of the central V domain of ALIX

severely attenuates budding in a dominant-negative manner (23) that depends on the

presence of the YP(X)nL motif. Recently, ALIX was found not only to bind to HIV-1 p6,

but also to the NC region of Pr55 via its N-terminal Bro1 domain (24-26), implicating a

possible cooperative or alternative function of NC for the recruitment of the ESCRT to

promote virus budding.

A third kind of L-domains consists of a PPxY motif and is found in Rous sarcoma virus

(RSV; (27)), murine leukemia virus (MLV) and more distantly related enveloped viruses

(8). The PPxY L-domain interacts with WW domains of the HECT (Homologous to E6-

associated protein C-terminus) family of E3 Ub ligases (28, 29). Recently, a member of

this family of Ub ligases, Nedd4-2s, has also been implicated in the egress of HIV-1 (30,

31).

In addition to the conserved retroviral assembly domains M, I and L, other regions

contribute to the efficient assembly and release of viral particles (for review see (32)),

amongst them the major homology region (MHR) in CA (33), the only region within Gag

that shows significant sequence homology between different retroviruses (34).

4.3 The ubiquitin proteasome system

Besides the lysosomal system, the ubiquitin proteasome system (UPS) is the major

proteolytic pathway in the cell (35). The 26S proteasome, a large, highly abundant multi-

Introduction 14

enzyme complex distributed throughout the cytosol and nucleus, is composed of the 20S

catalytic core particle (CP) and two regulatory 19S particles (RP).

The barrel-shaped CP consists of four stacked heteroheptameric rings, two outer α-rings

and two inner β-rings (α1-7, β1-7, β1-7, α1-7). The N-terminal Thr residues of the β1, β2

and β5 subunits represent the enzymatically active sites of the CP, acting as both

nucleophile and proton acceptor in hydrolysis. Proteasomes can cleave after most aa

residues, however, certain cleavage preferences have been attributed to the catalytic

subunits: the β1-subunit cleaves after acidic residues (caspase-like activity), the β2-

subunit after basic residues (tryptic activity), and the β5-subunit after hydrophobic

residues (chymotryptic activity; (36)).

According to its appearance in electron micrographs, the RP is subdivided into lid and

base. Within the 10-subunit base, a hexameric ring of ATPase subunits binds to the α-

subunits of the CP, mediates opening of the gate into the catalytic chamber, substrate

unfolding and translocation. Several subunits located both within the base and lid are

critical for the recognition of poly-Ub chains and substrate deubiquitination.

The immune-modulatory cytokine interferon (IFN)-γ induces the synthesis of the

immunosubunits β1i (low-molecular-weight protein 2 [LMP2]), β2i (multicatalytic

endopeptidase complex-like-1 [MECL1]) and β5i (LMP7) that can replace the

constitutive catalytic subunits in nascent 20S proteasomes to build the so-called

immunoproteasome (37, 38). However, immunoproteasomes are constitutively expressed

in some tissues like spleen, lymph node (LN), thymus and small intestine (39) and in

antigen presenting cells (APCs) like dendritic cells (DCs; (40)). The altered cleavage

specificities of immunoproteasomes is associated with differential antigen (Ag)

processing and results in a modified spectrum of major histocompatibility complex class I

(MHC-I)-presented epitopes (reviewed in (41)). In addition to catalytic immunosubunits,

IFN-γ induces the 11S activator, also named PA28, a ring-like structure composed of

three PA28α and two PA28β subunits (42), which enhances the activity of the CP (43-

47), probably by bringing the α-subunits in an open conformation to facilitate substrate

entry (42, 48).

Although Ub-independent pathways into the proteasome have been described (49),

substrates are in general targeted for proteasomal degradation by the covalent conjugation

of at least four Ub moieties in a process called polyubiquitinylation. After activation by

the activating enzyme E1, Ub is transferred onto an E2 conjugating enzyme and, by the

action of a specific E3 ligase, the C-terminus of Ub is attached via an isopeptide bond to

the ε-amino group of Lys residues of the target protein. Eukaryotic E3 ligases are divided

Introduction 15

into three classes according to their catalytic domain: HECT Ub ligases, ligases

containing a RING (really interesting new gene) domain and U-box E3 ligases. In

contrast to HECT E3 ligases, which form a thioester bond with the C-terminal Gly

residue of Ub before transfer to the target protein (50), RING-type E3 ligases serve as

platform that specifically recognizes and brings together the Ub-loaded E2 and the

substrate (51). The U-box was described as a modified RING finger domain and defines

the third group of E3 ligases (52, 53). In humans, two potential E1s, about 30 E2s and

over 600 E3s are encoded (54), with ~95 % of the latter being of RING-type. In addition,

E4 enzymes have been described, that can catalyze the extension of preexisting poly-Ub

chains already attached to the target protein (55, 56).

In a process called monoubiquitination, a single Ub molecule is attached to a Lys residue

of the target protein. This can also occur at several Lys acceptor sites, resulting in

multiple monoubiquitination, also named multiubiquitination. Within Ub itself, seven Lys

residues serve as potential acceptor sites for additional Ub moieties, resulting the

formation of poly-Ub chains (57). It is well established that the linkage to at least four

Lys48-linked Ub molecules (58) acts as a signal for recognition of the target protein by

subunits of the 19S RP of the 26S proteasome (58) or by adaptor proteins (59). The

substrate undergoes deubiqutination (60), allowing for Ub recycling, is funneled into the

CP, where it is degraded into peptides ranging from 4 to 25 residues.

Mono- and multiubiquitination as well as polyubiquitination linked via Lys63 have been

implicated in regulatory functions unrelated to proteasomal degradation governing for

example endocytosis of cell surface receptors, DNA-repair, signal transduction,

transcription and translation (61, 62). Recently, unconventional poly-Ub chains linked

through K6, K11, K27, K29 or K33 have been detected. Those are either linked to the

same Lys residue on each ubiquitin moiety to form a homogeneous chain, or to different

Lys residues to build a heterogeneous or branched chain. K11-linked poly-Ub chains

signal for proteasomal degradation, especially through ERAD (endoplasmic reticulum-

associated degradation (63)), and are involved in cell cycle regulation (64).

K29/K33-linked poly-Ub chains have been described to be assembled by HECT E3

ligases (65) and have been implicated in the UFD (Ub fusion degradation) pathway (66),

lysosomal degradation (67) and regulation of protein kinase activation (68). However, the

relevance of those and other unconventional poly-Ub chains in vivo is not fully

understood yet.

Introduction 16

Fig. 4.3: The ubiquitination cascade and types of ubiquitination. Ubiquitination starts with ATP-dependent formation of a thioester bond between the C-terminus of Ub and the E1 ubiquitin-activating enzyme, followed by transfer of Ub to an E2 ubiquitin-conjugating enzyme. Isopeptide bond formation between the C-terminal Gly residue of Ub and the ε-amino group of a Lys residue of the target protein is mediated by the action of E3 Ub ligases. RING-finger E3 ligases act as scaffolds, whereas HECT-domain E3s bind the activated Ub to a Cys residue and transfer it to the target protein. Ubiquitination can involve a single Ub (monoubiquitination) or multiple Ubs, each conjugated to a single Lys residue (multiubiquitination), both generally regulating protein function. Ubiquitination is reversible by the action of DUBs. As any of the seven Lys residues of Ub as well as its N-terminus can be acceptor sites for Ub attachment, poly-Ub chains in which Ub itself is successively ubiquitinated can be assembled. This might involve action a an E4 Ub chain elongation factor. Depending on the type of linkage, polyubiquitination can tag a substrate for proteasomal degradation or fulfill regulatory functions.

Introduction 17

In addition to Ub, so called Ub-like (UBL) proteins (so far, 17 of them have been

identified in mammalian cells), like SUMO (small Ub-related modifier), can also be

conjugated to proteins in a way that is rather similar to ubiquitination (69).

Like other processes that regulate protein function, for example phosphorylation,

ubiquitination is highly dynamic and reversible. The removal of Ub moieties is catalyzed

by deubiqutinating enzymes (DUBs), comprising almost 100 representatives in mammals

that are grouped into five families: Ub C-terminal hydrolases (UCHs), Ub-specific

proteases (USPs), ovarian-tumor (OTU) domain DUBs, Machado-Joseph domain (MJD)

DUBs and Jab1/MPN metalloenzyme (JAMM) zinc-dependent metalloproteases (for

review see (60, 70-72)).

Besides its numerous functions in cellular processes, the UPS plays an important role in

the replication cycle of viruses (73), amongst them HIV-1 (74-77). In particular, HIV-1

takes advantage of the UPS to facilitate replication first, by targeting cellular restriction

factors (78) for degradation by the proteasome through the action of accessory proteins

(79-84) and second, by utilizing the cellular endosomal sorting machinery, Ub ligases and

other components of the UPS for its egress (77). Moreover, Ag processing by the

proteasome generates the peptides that are presented by MHC-I molecules to CD8+ T

cells for the recognition and elimination of virus-infected cells (85, 86) and viruses have

evolved efficient strategies to down-modulate Ag presentation in order to evade immune

recognition (87).

4.4 Role of the UPS in late steps of HIV-1 replication

The observation that retrovirus particles are enriched in free Ub, made almost 20 years

ago (88), pointed towards a function of the UPS in retrovirus budding. The role of the

UPS for late steps of retrovirus replication was further emphasized by the finding that

inhibition of the proteasome resulted in a defect in release and maturation of HIV-1 and

other retroviruses that was highly reminiscent of the phenotype observed for L-domain

mutants (89-91). Intriguingly, not all retroviruses require proteasome activity for efficient

release. While the budding of HIV-1, HIV-2, MLV and MPMV, harboring PTAP- or

PPPY-type L-domains has been shown to be sensitive to proteasome inhibition, mouse

mammary tumor virus (MMTV) or EIAV with unknown, or YPDL L-domains,

respectively, did not show reduced virus release upon treatment with proteasome

inhibitors (PIs; reviewed in (9)). Although extensively studied (for review see (75, 77, 92-

97)), the function of the UPS in retrovirus release is still not fully understood.

Introduction 18

To catalyze the final abscission of the virus bud from the PM, HIV-1 and other

retroviruses usurp the cellular ESCRT machinery (92), which consists of five protein

complexes, ESCRT-0, -I, -II, -III, and Vps4-Vta1 as well as several ESCRT-associated

proteins (98-101). The ESCRT mediates the scission of membrane necks and is, besides

viral budding, involved in topologically highly similar processes, namely recognition and

sorting of monoubiquitinated proteins into endosomes (101), multivesicular body (MVB)

biogenesis (102, 103) and cytokinesis (104, 105).

Recent work has demonstrated that this abscission of membrane stalks is directly

mediated by ESCRT-III together with the AAA ATPase (ATPase associated with various

cellular activities) Vps4, both also representing the most evolutionary conserved

representatives of the ESCRT (106). The ESCRT-III subunits exist as soluble monomers

that polymerize into filaments, spirals or tubes that are tightly associated with

membranes, thereby causing membrane deformation (107-109). Recycling of the ESCRT-

III proteins back into their soluble monomeric form is accomplished by Vps4 in an ATP-

dependent fashion.

As described above, HIV-1 and other retroviruses harboring a PTAP L-domain motif

recruit the ESCRT machinery via binding to the ESCRT-I component Tsg101. Tsg101 in

turn interacts with ALIX, an ESCRT-III associated protein. Retroviruses containing a

YPDL motif on the other hand directly interact with ALIX to recruit ESCRT-III. Though

the ESCRT-II complex has been proposed to play a role in HIV-1 budding (19), it seems

to be dispensable for HIV-1 egress according to more recent findings (110). Thus, it

appears that HIV-1 and other retroviruses containing a PTAP L-domain utilize alternative

mechanisms to link ESCRT-I and III, thereby bypassing ESCRT-II.

As many components of the ESCRT bind to ubiquitinated cargo proteins (100), the

ubiquitination of retroviral Gag proteins (111, 112) has been suggested to play a role in

interaction with the ESCRT machinery (95). Two Lys residues within p6 have been

shown to be specifically mono-ubiquitinated (111), while one of them is also sumoylated

(113). The function of both modifications is, however, still unclear, as the Ub acceptor

sites within p6 seem to be dispensable for virus release and replication, Gag processing,

or incorporation of free Ub, at least in certain cell types (112). More recent observations

indicate that, although low-level monoubiquitination most likely occurs at multiple sites

within all Gag domains (114, 115), attachment of Ub to Lys-residues neighboring the

PTAP L-domain seems to be important for virus release and replication (115). Moreover,

there is evidence gained in vitro that monoubiquitination of p6 might influence the

binding properties of Tsg101 (116). Fusion of Ub to the C-terminus of RSV Gag is able

19

to rescue budding from PI-treated cells (89). Recently, it has been demonstrated that

fusion of Ub to the C-terminus of EIAV Gag can functionally replace the YPDL L-

domain (117). Replacement of the YPDL L-domain in EIAV Gag by a PPPY or a PTAP

L-domain rendered VLP release sensitive to PI treatment, while Ub fusion in this context

conferred resistance to proteasome inhibition. In the absence of a functional L-domain,

however, Ub fusion sensitized VLP release of EIAV to blockade of proteasome activity

(117). Therefore, different models have been proposed to explain the effect of PIs on

virus release. First, it has been suggested that PIs, by rapid depletion of free Ub, interfere

with the monoubiquitination of Gag, which serves as a recognition signal for the ESCRT

machinery. On the other hand, Ub fusion to EIAV Gag in absence of an L-domain confers

sensitivity to PI treatment, supporting a model in which proteasome shutdown disturbs

the cellular machinery required for particle release rather than directly affecting Gag

ubiquitination (117). Moreover, when cells are treated with PIs, inhibition of virus release

seems to occur faster than depletion of free Ub (Ulrich Schubert, unpublished observation

and (91))(9). In a distinct, third model, it has been proposed that proteasome inhibition

prevents the clearance of misfolded or otherwise damaged Gag molecules, which then

interfere in a dominant-negative manner with the ordered assembly of the Gag lattice

required for budding (89, 91).

While Gag ubiquitination has been shown to influence L-domain function, L-domains

themselves have been shown to either positively (118, 119) or negatively regulate the

ubiquitination of Gag (114, 120, 121), thus underlining the intimate relation between L-

domain function and the ubiquitination machinery.

Overexpression of the UBL protein ISG (IFN-stimulated gene) 15 blocks HIV-1 and

Ebola virus particle release (122-124). First reports indicated that ISG15 expression

inhibited the E3 ubiquitin ligase Nedd4 that interacts with the PPEY motif of Ebola virus

(124, 125) and prevented binding of Tsg101 to HIV-1 p6, though direct conjugation of

ISG15 to Tsg101 or Gag has not been demonstrated (123). Interestingly, ISG15

overexpression resulted in reduced levels of Gag ubiquitination (122, 123). Recently, it

has been proposed that ISG15 can additionally prevent the interaction of Vps4 with its

coactivator protein LIP5 due to conjugation of ISG15 to the ESCRT-III protein CHMP5

(122).

Introduction 20

4.5 Role of the UPS and defective ribosomal products (DRiPs) in

MHC-I antigen processing

The conventional MHC-I pathway starts with the processing of endogenous Ags by the

26S proteasome in the cytosol (85, 86, 126). It has now become widely accepted that the

majority of endogenous MHC-I ligands are derived from the processing of de novo

synthesized, dysfunctional proteins (reviewed in (127-131)). These so-called defective

ribosomal products (DRiPs; (132, 133)) fail to adopt their functional conformation and

are quickly eliminated by the UPS during or following translation in order to avoid cell

damage (134), regardless of the half-life of their native counterpart. Due to their rapid

turnover, DRiPs can only be detected in the absence of proteasomal activity (133, 135,

136), which can be easily achieved by the addition of cell-permeable PIs, for example the

irreversible and highly specific inhibitor lactacystin (137).

Endoplasmic reticulum (ER)-resident Ags can gain access to the cytosolic proteasome via

ERAD (138, 139). Ags that have not been synthesized by the APC itself, but taken up via

endocytosis, phagocytosis, macropinocytosis or even through gap junctions, can be

presented by MHC-I in a process called cross-presentation, that is especially crucial to the

priming of naïve CD8+ T cells by DCs (140-142). Recently, a process named autophagy,

in which intracellular material such as whole organelles, cytoplasmic material or protein

aggregates are sequestered into membrane-enclosed vacuoles, named autophagosomes,

and destroyed by fusion with lysosomes, has gained much interest in the field of

immunology. Autophagy not only provided an explanation for presentation of

endogenous peptides by MHC-II, but has also been implicated in MHC-I Ag presentation

(143-145).

Proteolysis by the 26S proteasome produces peptides three to 25 residues in length (146)

that are transported into the ER by TAP (transporter associated with antigen processing),

which is composed of TAP1 and TAP2 subunits (147) and has a specificity for peptides

ranging approximately from 7 to13 aa (148-150). In the ER, peptides undergo N-terminal

trimming by luminal proteases to fit into the peptide binding groove of MHC-I molecules,

which can bind, with only few exceptions, peptides 8-10 aa in length (151-153). Binding

is mostly mediated by the peptide’s so called anchor residues which have to be occupied

by specific residues depending on the haplotype of the MHC-I molecule. Folding of the

MHC-I α-chain (heavy chain) is mediated by the chaperone calnexin and the lectin-like

chaperone Calreticulin (CRT), followed by association with β2-microglobulin (β2m).

Introduction 21

Fig. 4.4: DRiPs as main source for epitopes presented via the conventional MHC-I pathway. 1) Protein de novo synthesis and quality control. Flawless proteins enter the standard protein life cycle, whereas defective ribosomal products (DRiPs) become rapidly polyubiquitinated. 2) Ag processing by the proteasome. 3) Peptide transport and trimming. The proteasome generates mainly N-terminally extended proteolytic intermediates that can be protected by the chaperone TRiC (tailless complex polypeptide-1 (TCP-1) ring complex) or CCT (chaperonin containing TCP-1), or destroyed by the protease TOP (Thimet oligopeptidase). In the ER, ERAAP (ER aminopeptidase associated with Ag processing) can N-terminally trim antigenic precursors imported by TAP. The cytosolic protease TPPII (tripeptidyl peptidase II) might contribute to Ag processing and/or trimming. 4) MHC-I folding and peptide loading. Nascent MHC-I are folded by the chaperones calnexin and CRT and undergo disulfide bond (S-S) formation and association with β2m. Fully folded MHC-I molecules are incorporated into the peptide loading complex (PLC), which catalyzes the loading with peptides imported from the cytoplasm by the heterodimeric TAP. Tapasin binds to TAP, MHC-I and the oxidoreductase ERp57. Tapasin and ERp57 are covalently linked by a disulfide bond. 5) Export, presentation and T-cell activation. MHC-I molecules loaded with high-affinity peptides are transported via the secretory pathway to the cell surface. Specific binding of the T-cell receptor (TCR) to a MHC-peptide-complex in presence of costimulatory signals (not depicted) triggers activation of CD8+ T cells.

The generation and stabilization of peptide-MHC-I (pMHC) complexes is accomplished

by a specialized ER-resident chaperoning complex, called the peptide-loading complex

(PLC), consisting of CRT, the thiol oxidoreductase ERp57, TAP and Tapasin, a

chaperone-like protein (154-156). Only peptide-loaded MHC-I molecules are transported

through the secretory pathway to the cell surface, where the pMHCs are presented to

CD8+ T cells.

Introduction 22

4.6 Regulation of UPS-mediated proteolysis by degradation signals

It has become increasingly clear over the last years that regulation of protein function and

degradation by the UPS plays a key role in a number of processes like cell cycle

progression, apoptosis, gene expression, stress responses, viral replication and MHC-I Ag

presentation. Defects in targeted proteolysis have been implied in the pathogenesis of

severe diseases, (157) including cancer (158, 159), autoimmunity (160), cardiovascular

disease (161) and neurodegeneration (162-164). The PI Bortezomib (also known as PS-

341 or Velcade®) has been approved in 2003 for the treatment of multiple myeloma (165-

167), a plasma cell malignancy that results in the overproduction of monoclonal

immunoglobulins, a feature that sensitizes multiple myeloma cells for PI-mediated

induction of apoptosis (168). Next generation PIs as well as strategies aiming at upstream

targets, like the development of DUB inhibitors or inhibitors of E3 ligases, are among

promising candidates for the treatment of various malignancies and other diseases (159,

169).

Given the extreme importance of tight control of protein half-life in the cell (134), it is

not surprising that there exist several motifs in the primary protein sequence, named

degrons (170), that target a protein for degradation by the 26S proteasome causing its

rapid turnover (171).

Many short-lived proteins contain regions rich in Pro (P), Glu (E), Ser (S) and Thr (T),

which were identified by correlation of the metabolic half-life with the primary sequence

of proteins in silico and named PEST-sequences (172). Removal of PEST sequences from

short-lived proteins markedly augmented metabolic stability (173-176), thus supporting

the functionality of PEST sequences in vivo. Likewise, transfer of a PEST sequence to

otherwise stable proteins can induce rapid turnover (177, 178).

The so called N-end rule, which relates the metabolic half-life of a protein to the identity

of its N-terminal aa has been first defined by A. Varshavsky in yeast (179) as another

principle that governs protein stability. The entire degradation signal, the N-degron,

consists of the destabilizing N-terminal aa, grouped into basic (Arg, Lys, His) and

hydrophobic (Leu, Phe, Trp, Tyr, Ile) residues, and an internal Lys residue (180). Many

components of N-end rule pathway have been identified in pioneer studies using

engineered model substrates like β-Galactosidase (β-Gal; (179-182)). More recently,

however, a number of physiological substrates and their functions as well as the enzymes

that create and recognize N-degrons have been characterized (183-187).

23

The N-terminal initiator Met residue can be cotranslationally removed by Met

aminopeptidases (MetAPs), but only if the following residue possesses a small side chain

(Gly, Val, Pro, Ala, Ser, Thr or Cys; (188-190)). As primary destabilizing residues cannot

be created this way, further modification is necessary. If the exposed residue is a Cys,

however, it can be first oxidized by molecular oxygen or nitric oxide (NO) and then

arginylated by an enzyme called arginyl-tRNA-protein transferase (Ate1; (191, 192)),

which has been found to be important for the degradation of several regulator of G-

protein signaling (RGS) proteins (183, 186, 193-196).

Arg, a primary destabilizing residue, can also be attached by Ate1 to proteins that expose

Asp or Glu, which can in turn be created from Asn or Gln by the action of N-terminal

amidases (NTAs; (197, 198)). N-end degrons can also be exposed by endoproteolytic

cleavage. This mechanism has been demonstrated for the first physiological substrate of

the N-end rule pathway in yeast, Scc1, a subunit of the cohesion complex, which is

important for chromosome stability (199). Degradation by the N-end rule pathway

following proteolytic cleavage has also been observed for the HIV-1 Int (200), the

Sindbis virus RNA polymerase (201) and Drosophila inhibitor of apoptosis (DIAP1

(185)). In this way, substrates of the N-end rule pathway can be engineered by N-terminal

in frame fusion with Ub that is co-translationally cleaved by DUBs (71, 202).

The E3 Ub ligases that recognize N-degrons, named N-recognins, have been identified as

a family of proteins containing an approximately 70 aa zinc finger-like domain, called the

UBR box. In mammals, at least seven UBR box proteins have been described (203),

whereas in yeast, recognition of N-end rule substrates is achieved by binding of a single

N-recognin, the UBR1 gene product, to primary destabilizing residues (204).

Cleavage of engineered Ub fusions by DUBs is abolished, if the C-terminal Gly residue

of the Ub moiety is exchanged for a different aa, for example Ala or Val. Such fusion

proteins are substrates for the so called Ub-fusion degradation (UFD) pathway (66, 205)

which includes polyubiquitinylation of the Ub moiety itself at Lys48 or Lys29 (66), and

proteasomal degradation of the fusion protein.

Other motifs of the protein primary sequence that govern protein stability include

phosphodegrons in cyclins or an oxygen-dependent degron (ODD) in hypoxia-inducible

factor-1α (HIF-1α; for review see (171)).

Clearance of damaged proteins, including misfolded or mistargeted polypeptides is one of

the crucial functions of the UPS. It has been shown that mistargeting can lead to rapid

degradation of a protein, thus demonstrating that the correct subcellular localization

represents an essential quality criterion (206, 207), as is correct folding (208-210).

Introduction 24

4.7 Correlation between metabolic half-life and MHC-I antigen

presentation

The correlation between Ag stability and generation of MHC-I antigenic determinants has

been extensively studied (207, 210-217). First, it has been demonstrated in a recombinant

vaccinia virus (rVV) system that expression of an instable variant of the influenza A virus

(IAV) nucleoprotein (NP) increased the efficiency in presentation of one particular

epitope of NP (207), a finding that has subsequently been extended by others (211, 212).

Recombinant Ub-X-Gal fusion proteins were differentially degraded according to the N-

end rule in an Ub- and proteasome-dependent manner when introduced exogenously into

cells and the instable counterparts of these fusion proteins evoked enhanced T-cell

activation (213). A correlation between protein half-life and generation of class-I ligands

has also been reported for the listeria Ag p60 (214, 215). In contrast, no such correlation

has been found for generation of the SIINFEKL (SL) epitope derived from instable

Ovalbumin (OVA) and β-Gal fusion proteins (217). Enhanced Ag presentation together

with efficient stimulation of cytotoxic T lymphocytes (CTLs) has been shown for a

metabolically instable variant of the HIV-1 accessory protein Nef (216). However,

instable variants of the HIV-1 Gag polyprotein failed to elicit enhanced CTL responses

although increased numbers of Gag-derived epitopes were presented on the cell surface

(210).

To investigate the correlation between efficiency of protein degradation and MHC-I Ag

presentation, the HIV-1 Gag polyprotein has been chosen as a model Ag for two reasons:

First, it has already been demonstrated that Gag as a viral Ag can enter the DRiP-pathway

(133) and, second, Gag represents a highly attractive Ag for vaccine development. Using

the OVA-derived SL peptide as a model epitope, the efficiency of entry of Gag variants

carrying certain degradation signals into the MHC-I pathway was investigated.

Gag was expressed from a codon-optimized, synthetic gag gene, which results in high

expression levels and efficient VLP release and allows studying the properties of Pr55 in

the absence of other HIV-1 proteins (218). The impact of degradation signals on

synthesis, metabolic stability, budding as well as localization of HIV-1 Pr55 was

analysed.

In addition, the PTAP L-domain motif was found not only to function in HIV-1 budding,

but also in the regulation of MHC-I Ag presentation of HIV-1 Gag.

Results 25

5 Results

5.1 Targeting HIV-1 Gag into the DRiP-pathway enhances MHC-I

antigen presentation and CD8+ T-cell activation

5.1.1 Construction of Gag variants containing degradation signals

Although the correlation between protein half-life and MHC-I Ag presentation has been

demonstrated for a number of Ags (207, 210-217), the role of protein degradation motifs

in targeting Ags to the DRiP pathway is not well studied. As Gag is considered an

attractive target for vaccine development (219-221), it was a central aim of this thesis to

enhance the generation of Gag-derived epitopes for MHC-I presentation by targeting the

Gag polyprotein Pr55 originating from the isolate HIV-1HX10 (named Gag from hereon)

for rapid degradation by the 26S proteasome. Assuming the importance of DRiPs as the

major source of antigenic peptides, it was hypothesized that introduction of degradation

signals attracts Gag into the DRiP-pathway.

To circumvent the necessity for co-expression of the regulatory HIV-1 tat and rev genes,

which are strictly required for efficient transcription (222) and nuclear export of Gag-

specific mRNA (223), respectively, a codon-optimized, synthetic gag gene was expressed

under the control of the cytomegalovirus (CMV) promoter, resulting in high and Rev-

independent expression levels of Gag (synGag) in various cell lines (218).

A schematic representation of the Gag variants, most of which were kindly provided by

Prof. Ralf Wagner (Institute of Medical Microbiology and Hygiene, University of

Regensburg) is shown in Fig. 5.1. A putative N-end rule substrate was constructed by N-

terminal in frame fusion to Ub (UbRGag). This results in the expression of an Arg as

destabilizing N-terminal aa (RGag) after cotranslational removal of the Ub moiety by

DUBs that cleave with high efficiency between the C-terminal Gly of Ub and all aa

except Pro (224). The fusion UbMGag, corresponding to the unmodified wt Gag protein

upon Ub removal, was created as a control. The C-terminal PEST sequence derived from

the murine ornithine decarboxylase was inserted in frame to the C-terminus of Gag. To

possibly induce misfolding of Gag, single aa substitutions were introduced in the N-

terminal region (P231L) as well as the MHR (L304H) of CA.

Mistargeting of Gag was induced by mutation of the N-terminal myristoylation site

(G2A). In addition, a putative substrate of the UFD pathway was constructed by mutation

of the C-terminal Gly residue of the Ub moiety (UbG76VGag).

Results 26

Fig. 5.1: (A) Schematic representation of Gag variants containing degradation signals. (B) Insertion of the SL epitope into the p2 spacer of Gag is depicted. Amino acids are indicated in single letter code.

5.1.2 Introduction of the OVA-derived SL epitope as indicator for Ag processing of

Gag

Unfortunately, there have been no TCR-like Abs described to date which recognize

MHC-I-bound Gag-derived epitopes. As isolation and handling of Gag-specific CTL

clones derived from HIV-1-infected patients is rather difficult, the OVA-derived SL

model epitope of was introduced as an indicator for Ag processing into the p2 spacer

region localized between the CA and NC domains of Gag (Fig. 5.1 B; GagSLp2). To avoid

interference with the structure and function of the p2 spacer, the SL epitope was

introduced C-terminally of a putative α-helix spanning the C-terminal domain of CA and

the N-terminal region of p2 (225, 226). Moreover, there is a certain length polymorphism

in this region between HIV-1 isolates (www.hiv.lanl.gov). This strategy allows for the

quantification of the number of SL epitopes derived from the Ag processing of Gag using

the mAb 25D1.16, which specifically recognizes the SL epitope bound to the murine

MHC-I molecule H2-Kb (227). As the 25D1.16 Ab selectively binds to SL in complex

Results 27

with H2-Kb, it was necessary to use either H2-Kb-positive murine cell lines or H2-Kb-

transgenic human cell lines for the analysis of SL-presentation. For a first set of

experiments, the murine thymoma cell line EL4 that naturally expresses H2-Kb was

selected to ensure that SL-presentation was analysed in the context of physiological H2-

Kb expression levels.

5.1.3 Generation and characterization of GagSL-expressing EL4 cell lines

In contrast to human cell lines, in which high expression levels of Gag were obtained

after transient transfection with psyngag expression plasmids (see Fig. 5.8), Gag or

GagSL proteins were almost undetectable by Western blotting or flow cytometry after

transient transfection of murine EL4 cells (data not shown). To obtain sufficient numbers

of Gag-positive cells for FACS analysis of Ag presentation, EL4 cells were stably

transfected with Gag expression constructs. When bulk EL4 cell populations were

analysed for presentation of the SL epitope using the mAb 25D1.16, an enhanced SL-

presentation was reproducibly detectable on the surface of cells expressing Gag variants

harboring certain degradation signals (performed by Dr. A. Goldwich, data not shown).

This was especially observed for the N-end rule substrate UbRGagSL, which was further

compared to its counterpart UbMGagSL, expressing N-terminally Met instead of

destabilizing residue Arg (see Fig. 5.1). After single cell cloning and several passaging

steps, two stable clones of transgenic EL4 cells which stably express the UbMGagSL and

UbRGagSL proteins, respectively, were selected.

Western blot analysis using anti-p24 and anti-p6 antibodies revealed that Gag and GagSL

proteins migrated at the appropriate MW of approximately 55 kDa confirming the

complete cleavage of the Ub moiety (Fig. 5.2). In addition to the 55 kDa Gag signal, a

second band migrating at approximately 40 kDa was observed that possibly results from

cleavage of Gag by a cellular protease. A slightly slower migration behavior of GagSL

proteins reflects the presence of the SL epitope (Fig. 5.2). In addition, cell extracts

standardized for protein content were analysed by p24-ELISA to determine the

intracellular steady-state level of Gag (performed by Dr. A. Goldwich, data not shown).

The level of Gag in the UbMGagSL/EL4 clone (4.14±2.36 ng of p24/106 cells) was

almost twice the amount of Gag determined in the UbRGagSL/EL4 clone (1.59±1.14 ng

of p24/106 cells). This slight difference in Gag expression was repeatedly observed,

indicating that the lower steady-state level of Gag in the UbRGagSL/EL4 clone represents

an inherent characteristic of the N-end rule substrate.

Results 28

Fig. 5.2: Analysis of Gag expression in clonal EL4 cell lines by Western blotting. Similar amounts of whole cellular protein were subjected to SDS-PAGE and Western blotting using anti-p6 or anti-p24 antiserum. Parental EL4 cells or EL4 cells expressing the SL epitope from a minigene construct served as negative controls. As a loading control, blots were reprobed using an anti-β-actin Ab.

5.1.4 Half-life and DRiP-rate of UbRGagSL and UbMGagSL proteins

To determine the overall protein half-life, pulse radiolabeling of EL4 cells with [35S]Met

for 20 min followed by a 48 h long chase was performed. Gag proteins were recovered by

IP using Gag-specific Abs, subjected to SDS-PAGE and visualized by autoradiography or

phosphorimaging (Fig. 5.3 A). In parallel, total cell lysates were resolved by SDS-PAGE

and the major band corresponding to β-actin served as an internal control (Fig. 5.3 A,

lower panel). Densitometric analysis of three independently performed pulse-chase

experiments revealed that both proteins displayed an overall half-life of approximately six

hours in EL4 cells, though the decline of Gag expressed in the UbRGagSL/EL4 clone was

somewhat faster during the first 24 h of chase when compared with the UbMGagSL/EL4

clone (Fig. 5.3 B). This slightly reduced half-life together with the generally somewhat

reduced steady-state level of the UbRGag protein (Fig. 5.2) might point towards an

increased turnover occurring in concert with de novo synthesis.

Due to the extremely delicate nature of DRiPs as short-lived products which are at least

partially ubiquitinated and degraded by the 26S proteasome, detection can only be

achieved after shutdown of proteasome activity and blocking deubiquitinating enzyme

activities (133). To determine the DRiP-rate of UbM- and UbRGagSL proteins,

respectively, short-term pulse-chase experiments were conducted according to previously

elaborated DRiP pulse-chase protocols (133, 135). Gag-transgenic EL4 cells were treated

during the final 10 min of a 30 min starvation period with a combination of distinctly

acting proteasome inhibitors, the peptide aldehyde zLLL (228) and the irreversibly and

highly specifically acting inhibitor lactacystin (LC; (137)).

Results 29

Fig. 5.3: Pulse-chase analyses of UbMGagSL/EL4 and UbRGagSL/EL4 cells. (A+B) Half-life of UbGagSL proteins. For long-term pulse-chase experiments, UbMGagSL/EL4 and UbRGagSL/EL4 cells were radiolabelled for 20 min with [35S]Met and chased for up to 48 h. (A) Fluorograph of Gag proteins recovered by IP using anti-p6 and anti-p24 antibodies and separated by SDS-PAGE. The band corresponding to β-actin was identified based on its MW in fluorographs of total cell lysates resolved by SDS-PAGE. (B) Densitometric quantification of [35S]-labeled Pr55. The radioactivity of the Pr55 band was quantitated using a phosphor imager and plotted as percentage of the initial signal. Values represent the mean and standard deviation (SD) from three independent pulse-chase experiments. (C+D) DRiP-rate of UbGagSL proteins. For short-term pulse-chase experiments, UbMGagSL/EL4 and UbRGagSL/EL4 cells treated with 20 µM of zLLL/LC each during the final 10 min of a 30 min starvation were radiolabelled for 15 min and chased for up to 60 min the presence or absence of zLLL/LC. (C) Fluorograph of Gag proteins recovered by IP and resolved by SDS-PAGE as described above. (D) Densitometric quantification of [35S]-labeled Pr55 (upper panels) and the high MW (HMW) smear recovered with anti-Gag antibodies (lower panels). The radioactivity recovered at each time point is plotted as percent of the radioactivity recovered at the time point “0” in absence of PIs. Mean values and SD from three independent pulse-chase experiments are depicted.

Cells were pulsed with [35S]Met for 15 min and chased for up to 120 min in the presence

or absence of zLLL/LC. Soluble Gag proteins in the detergent cell extracts that bound to

Gag-specific Abs were subjected to SDS-PAGE and analyzed by fluorography

Results 30

(Fig.5.3 B). The quantities of radioactivity in dried gels corresponding either to Pr55 or

the total proteins migrating in the high molecular weight (HMW) range of ~60-250 kDa

were quantitated using phosphorimaging instrumentation (Fig. 5.3 D). Similar to the long-

term pulse-chase (Fig. 5.3 A+B), the decay of Gag was slightly faster in UbRGagSL/EL4

cells (Fig. 5.3 C+D). While data obtained in both cell lines with active proteasomes were

comparable, the situation after proteasome shutdown was quite different: although

addition of zLLL/LC rescued Gag proteins in both cultures from degradation, the amount

of Pr55 recovered from UbRGagSL/EL4 cells increased by more than 50 % immediately

after the pulse, reaching maximum values of up to 80 % increase within 15 min

(Fig. 5.3 C, upper right panel) compared to only 30 % increase of Pr55 recovered from

UbMGagSL/EL4 cell extracts (Fig. 5.3 C, upper left panel). The same effect was

observed for the Gag fragment migrating at ~40 kDa: the rate of recovery of which was

again more pronounced in UbRGagSL/EL4 cells when compared to UbMGagSL/EL4

cells. In addition, an increase in the smear of proteins migrating in the HMW range was

detected in cells treated with zLLL/LC. This smear recovered by anti-Gag antibodies was

not recovered under the same conditions from non-transfected EL4 cells (data not

shown), indicating that the smear represents polyubiquitinated Gag-DRiPs and not

cellular proteins that bind non-specifically to beads or Gag-anti-Gag immune complexes.

Similar to previous calculations in HIV-1 expressing cells (133), the radioactivity in this

MW range was taken into account for the estimation of the DRiP-rates of both UbGagSL

fusion proteins. Though treatment of UbMGagSL/EL4 cells with zLLL/LC resulted in

modestly enhanced recovery of proteins in the HMW range, the magnitude of increase

was clearly higher in UbRGagSL/EL4 cells (Fig. 5.3 D, lower panels), confirming results

observed for Pr55. Similar to the kinetic of DRiP formation reported previously (133), the

accumulation of labeled Gag proteins during the pulse and approximately the first 15 min

of chase is of transient nature (Fig. 5.3 C+D). After reaching a certain plateau, processes

like deubiquitination, proteolysis or aggregation, as well as association with membranes

(114, 121), altogether continuously remove DRiPs from the pool of total Gag proteins

accessible for IP. Thus, the results of the pulse-chase analyses demonstrate that the DRiP-

rate of the Gag proteins clearly followed the N-end rule although the N-terminal fusion of

UbR to Gag does not result in a significant reduction of the overall metabolic half-life.

Results 31

5.1.5 Correlation of DRiP-rate with the MHC-I presentation of Gag-derived SL

Although the overall stability of both Gag proteins was not governed by the N-end rule,

an observation which is consistent with previous reports by others using different Gag

expression systems (210), an elevated DRiP-rate of the UbRGagSL variant compared to

UbMGagSL was observed. To analyze of whether UbRGagSL enters the MHC-I

processing pathway more efficiently, the presentation of SL in complex with H2-Kb

molecules was assessed by flow cytometry using the mAb 25D1.16 conjugated to Alexa

Fluor 647 (25D1.16-647). In parallel, the expression of Gag was monitored by

intracellular staining with a CA-specific Ab (KC57-FITC). Parental EL4 cells and a cell

clone expressing relatively high levels of the SL epitope from a minigene construct,

called SL/EL4, served as negative and positive controls, respectively. Similar to previous

studies using rVV expression systems (211, 229, 230), a CMV-promoter construct that

directs high level expression of the sequence MSIINFEKL was used to generate the

stably transfected SL/EL4 clone.

Fig. 5.4: Flow cytometry analyses of SL-presentation on the surface of the UbRGagSL/EL4 and the UbMGagSL/EL4 cell lines. (A) Histogram plot of UbMGagSL/EL4 and UbRGagSL/EL4 cells double-stained for SL-H2-Kb complexes at the cell surface using the mAb 25D1.16 conjugated to AlexaFluor64 (25D) and intracellular Gag using a CA-specific mAb (KC57-FITC) (B) Quantification of six independent experiments after normalization of the mean fluorescence intensity (MFI) of the 25D1.16-647 staining to the MFI of the KC57-FITC staining. Parental EL4 cells or EL4 cells expressing the SL epitope from a minigene construct served as negative control and positive control, respectively. Bars represent mean values +/- SD (n=6; * = p<0.05).

FACS analyses revealed that the cell surface presentation of the SL epitope generated

from UbRGagSL clearly surpasses the level of presentation observed for the UbMGagSL

counterpart (Fig. 5.4 A). However, consistent with the ELISA and Western blot data, the

Results 32

level of Gag in UbRGagSL expressing cells is about half of that observed in the

UbMGagSL transgenic cells as shown by double staining with 25D1.16-647 and KC57-

FITC (data not shown). To further support the notion that the increased presentation of

H2-Kb-SL complexes on the surface of UbRGagSL/EL4 cells is not simply dependent on

variations in expression levels of Gag, the number of H2-Kb-SL complexes presented at

the cell surface was correlated with the intracellular amount of Gag in six independently

performed double-staining experiments (Fig. 5.4 B). The ratio of the mean fluorescence

intensity (MFI) at 647 nm (staining with 25D1.16-647) and the MFI at 488 nm (staining

with KC57-FITC) was calculated, demonstrating an approximately two-fold, significant

increase in SL-presentation.

Fig. 5.5: Analysis of loading of H2-Kb with SL epitopes. UbMGagSL/EL4 and UbRGagSL/EL4 cells were incubated at pH 3 for 2 min to dissociate Kb-bound peptides. After neutralization (0 min), cells were cultured for indicated time periods without inhibitors (A+B) or in the presence of PI (20 µM of zLLL) or a cocktail of protein biosynthesis inhibitors (PSIs) (C+D). The amount of surface H2-Kb (A+C) or H2-Kb-bound SL epitope (B+D) was analysed by flow cytometry. Data represent one of three independent experiments.

If the main source of SL epitopes are short lived Gag-DRiPs, the processing of

UbRGagSL should lead to a higher intracellular steady-state level of the SL epitope

compared to UbMGagSL. This should result in more efficient loading of empty H2-Kb

molecules with SL epitopes. To challenge this assumption, cells were subjected to a

standard acid wash procedure causing the dissociation of pMHC complexes and

subsequent decay of MHC-I heterodimers on the cell surface (212, 231). The fate of total

Results 33

H2-Kb molecules in both UbMGagSL/EL4 and UbRGagSL/EL4 cell clones after the acid

wash (time point “0 min”¸ Fig. 5.5 A) was comparable and revealed a reduction below

50 % of the original value (time point “-10 min”; Fig. 5.5 A). After the acid wash a slow,

but continuous recovery of cell surface H2-Kb molecules was observed that followed

comparable kinetics in both cell clones (Fig. 5.5 A). Although both, the steady-state

levels and the kinetics of recovery of H2-Kb, were almost identical in both cell clones,

specific staining of SL-H2-Kb complexes revealed a different picture: first, and as

observed already above (Fig. 5.4), the presentation of SL epitopes was approximately

twofold higher in UbRGagSL processing cells (Fig. 5.5 B, time point “-10 min”). Second,

after acid wash that causes a drop to almost identical baseline levels (Fig. 5.5 B, time

point “0 min”), the kinetic of recovery of SL-H2-Kb complexes on the cell surface was

clearly faster in the UbRGagSL/EL4 clone. According to previous studies that

demonstrated the generation of epitopes from de novo synthesized Ag of different origin

in a proteasome dependent manner (133, 211, 228, 232-236) control experiments using

protein synthesis inhibitors (PSIs) or PIs (Fig. 5.5 C and D) revealed that both, protein

biosynthesis as well as the activity of the proteasome are necessary in both cell clones for

efficient MHC-I Ag presentation. This was shown for total H2-Kb (Fig. 5.5 C) as well as

SL-loaded H2-Kb molecules (Fig. 5.5 D).

5.1.6 In vitro activation of the SL-H2-Kb specific T-cell hybridoma B3Z

In order to analyze whether the augmented SL-presentation on UbRGagSL/EL4 cells

compared to UbMGagSL/EL4 cells results in enhanced T-cell activation, the SL-H2-Kb-

specific T cell line B3Z was used. The B3Z hybridoma cell line has been generated by

fusion of the NFAT-lacZ T cell clone with a SL-specific Vα2/Vβ5 T cell clone (237) and

expresses the lacZ gene under control of the NFAT promoter. After overnight co-

cultivation of B3Z cells with corresponding EL4 target cells, the specific T-cell activation

was detected by a colorimetric assay based on β-Gal activity, thus detecting TCR

signaling.

First, we established that the experimental conditions were adequate to detect specifically

differences in the presentation of H2-Kb-SL complexes. Incubation of B3Z cells with

increasing concentration of the synthetic peptide SIINFEKL or a control peptide

SIIKFEKL demonstrated that exogenously added epitopes are bound to cell surface H2-

Kb molecules and presented to B3Z cells (Fig. 5.6 A). A linear correlation between

peptide concentration and activation of B3Z cells was observed in the range of 0.3 to

Results 34

30 ng/ml, which corresponds to approximately 0.3 to 30 nM of available peptides.

Complete absence of T-cell activation in the case of the control peptide confirms the high

specificity of the B3Z line. In a further experiment, parental EL4 cells were incubated for

1 h with increasing concentrations of exogenously added peptides. After intensive

washing, the cells were mixed with B3Z cells in an E (effector):T (target) ratio of 1:1

(Fig. 5.6 B), revealing that even a short peptide pulse can induce T-cell activation that

started at 10 ng/ml of added SL. Thus, the B3Z cell line represents a very sensitive and

specific indicator for SL-presentation which is able to detect subtle differences in epitope

density at the level of the signaling pathway. When Gag expressing EL4 clones were co-

cultivated with B3Z cells at different E:T ratios ranging from 1:10 down to 1:0.013, a

significant stronger T-cell activation was observed for the UbRGagSL/EL4 clone

compared to the UbMGagSL/EL4 counterpart, as shown in a representative experiment in

Fig 5.6 C.

Fig. 5.6: In vitro activation of the SL-H2-Kb-specific T cell line B3Z. (A) B3Z cells were incubated with the synthetic peptides SIINFEKL or SIIKFEKL in rising concentrations and activation of B3Z cells was assessed. (B) Parental EL4 cells were incubated for 1 h with externally added peptides in various concentrations. After intensive washing, the cells were co-cultured with B3Z cells in an E:T ratio of 1:1. (C) Transgenic EL4 cells were co-cultured with B3Z cells in an T:E ratio ranging from 10:1 to 0.013:1 in 96 well plates for 16 h. T-cell activation was analyzed by addition of 0.15 mM CPRG/0.5 % NP-40 in PBS and measuring the absorbance at 595 nm. AUFS: arbitrary units full scale.

Results 35

5.1.7 In vivo activation of SL-H2-Kb-specific OT-1 cells and induction of SL-

specific CD8+ T cells in naïve mice

To test whether the enhanced MHC-I presentation of SL epitopes generated from the

UbRGagSL variant also augments T-cell stimulation in vivo, adoptive transfer

experiments in which UbMGagSL/EL4 and UbRGagSL/EL4 cells were transferred

together with OT-1 splenocytes into naïve C57BL/6 mice were performed by Dr. A.

Goldwich. The OT-1 mouse is transgenic for the SL-specific TCR (Vα2/Vβ5), with

approximately one third of all splenocytes as well as LN cells representing naïve, SL-

specific CD8+ T cells (238). As positive controls both, the OVA-expressing EL4-derived

cell line E.G7 (239) as well as the minigene-expressing SL/EL4 cell clone (see

Fig. 5.4 A) were employed, while the parental EL4 cells served as negative control. To

quantify proliferation of OT-1 T cells, splenocytes and LN cells derived from OT-1 mice

were labeled with CFSE (Carboxyfluorescein succinimidyl ester). This method is meant

to track the number cell divisions in vivo (240). In order to prevent target and effector cell

encounter prior to injection and, in addition, to enhance the chance of interaction of both

cell types in the recipient mouse, OT-1 cells were first injected into one tail vein and,

after a delay of 5 min, the SL-presenting EL4 cells were injected into the contralateral

vein. Two days following cell transfer, the spleens of the recipient C57BL/6 mice were

removed and the splenocytes were analyzed by flow cytometry. Among splenocytes, the

OT-1 cells were identified as Vα2- and CFSE-positive cells (Fig. 5.7 A, gate R3). In

contrast to Vα2-negative cells (Fig. 5.7 A, gate R4), a certain fraction of the Vα2-positive

cell population showed clear evidence for proliferation which was evident from a

decaying CFSE fluorescence intensity. Analyzing Vα2- and CFSE-positive cells derived

from the UbRGagSL/EL4 recipient revealed a relatively higher number of OT-1 cells that

were primed and had started to proliferate compared to OT-1 cells derived from the

UbMGagSL/EL4 recipient mice (Fig. 5.7 A, lower panels). This notion was further

supported by the down-regulation of CD62L-expression on the surface of Vα2- and

CFSE-positive cells which is indicative of an activated T cell phenotype (data not shown).

The statistical analysis of 10 independent experiments revealed that SL-expressing cells

stimulate the Vα2- and CFSE-positive population of the transferred spleen and LN cells

to proliferate at different levels (Fig. 5.7 B). To compare differences in OT-1 T-cell

stimulation, a so-called proliferation index was calculated, which takes into account the

number of proliferating cells relative to the number of cell divisions.

Results 36

Fig. 5.7: T-cell activation by UbMGagSL/EL4 or UbRGagSL/EL4 cells in vivo. (A+B) Activation of OT-1 cells (A) Two days after adoptive co-transfer of UbM- or UbRGagSL/EL4 cells and CFSE-labeled OT-1 cells into wt C57BL/6 mice, splenocytes were analysed by flow cytometry. The CFSE-signal of Vα2-positive OT-1 cells in the live lymphocytes-gate was analyzed. Plots represent data from one out of ten mice. (B) To calculate the proliferation stimulus of the transgenic EL4 cells, the cell numbers of primed relative to naïve Vα2-positive cells (nprimed/nnaïve) was multiplied by the number of cell divisions (log2(MFInaïve/MFIprimed)). Values are given as mean ± SD (n=10, *=p<0.05). (C) Quantification of the SL-specific T-cell response by IFN-γ ELISPOT after immunization of naïve mice with EL4 cells. C57BL/6 mice were intravenously injected with UbMGagSL/EL4 or UbRGagSL/EL4 cells, respectively. Parental EL4 cells served as negative, synthetic SL peptide injected subcutaneously (SL s.c.) as positive control. Splenocytes were isolated 9 days post immunization and incubated with or without synthetic SL peptide. Each circle represents the frequency of IFN-γ secreting T cells from one individual mouse (mean of triplets). Mean values are depicted as bars (n = 14, *=p<0.05).

37

The comparison of the proliferation indices induced by UbRGagSL/EL4 cells (0.85+/-

0.31) and UbMGagSL/EL4 cells (0.53+/-0.25) revealed that UbRGagSL induces a

significantly stronger proliferation of adoptively transferred OT-1 T cells. Thus, the

efficiency of SL-presentation in UbRGagSL/EL4 cells correlates with a better T-cell

activation in vivo as quantified on the level of T-cell proliferation.

To further substantiate the notion that the increased DRiP-rate and MHC-I Ag

presentation correlate with a more efficient induction of a T-cell response in vivo, naïve

C57BL/6 mice were injected intravenously with UbMGagSL/EL4 or UbRGagSL/EL4

cells, respectively, and activation of SL-specific CD8+ T cells was quantified by IFN-γ

ELISPOT. Parental EL4 cells served as negative control, while synthetic SL peptide

(50 µg) was injected subcutaneously (SL s.c.) as a positive control. The results shown in

Fig. 5.7 C reveal that significantly higher frequencies of SL-specific T cells were induced

by immunization with UbRGagSL/EL4 cells when compared to immunization with

UbRGagSL/EL4 cells. These data indicate that, in naïve mice, UbRGagSL/EL4 cells are

more potent in activating not only transgenic OT-1 T cells, but also naïve SL-specific T

cells.

5.1.8 In human cells, Gag is targeted into the MHC-I pathway by the N-end rule,

but even more efficiently by stable N-terminal fusion to Ub

In parallel to experiments performed in murine EL4 cells, the number of SL epitopes

derived from the Ag processing of UbMGagSL or UbRGagSL was assessed by flow

cytometry in a human cell line. In contrast to the low expression levels obtained in murine

cell lines (Fig. 5.2 and data not shown), high expression levels were achieved after

transient transfection of the human cell lines HeLa or 293T with psyngag expression

plasmids (Fig. 5.8 and data not shown), allowing for FACS analysis of bulk cell

populations.

Following transient transfection with psyngag expression plasmids, transgenic HeLa cells

that stably express high levels of H2-Kb (see Fig. 5.14), named HeLa-Kb (241), were co-

stained with 25D1.16-647 specific for the H2-Kb-bound SL epitope and, intracellularly,

with KC57-FITC for detection of GagSL. To normalize for possible differences in Gag

expression levels, the MFI of the staining with 25D1.16-647 was divided by the MFI of

the staining with KC57-FITC after gating on Gag-expressing cells. Cells expressing Gag

lacking the SL sequence served as a negative control for the 25D1.16 staining. The data

of four independently performed experiments are shown in Fig. 5.8 A.

Results 38

Fig. 5.8: Analysis of SL-presentation in transiently transfected HeLa-Kb cells. (A) Comparison of the presentation of the SL-epitope inserted at the N-terminus of Gag (SLN-term), in the p2 spacer region (SLp2) or at the C-terminus of Gag (SLC-term). Following transfection of HeLa-Kb cells with expression plasmids coding for GagSL variants, H2-Kb-SL complexes presented on the surface of Gag-positive cells were quantified by flow cytometry using 25D1.16-647. Cells expressing wt Gag lacking the SL epitope served as a negative control. Bars represent mean values +/- SD (n=4; * = p<0.05; ns = not significant). (B) A fraction of the transfected HeLa-Kb cells analysed in A was lysed and, together with VLPs pelleted from the cell culture supernatant, subjected to SDS-PAGE and Western blotting using anti-p24 antiserum. As loading control, membranes were reprobed using an anti-β-actin Ab.

Compared to wt GagSL, higher numbers of SL-H2-Kb complexes were detected on the

surface of HeLa-Kb cells expressing UbMGagSL. Expression of UbRGagSL resulted in a

further increase in SL-presentation, albeit not statistically significant. Stable N-terminal in

frame fusion of Gag to Ub that is achieved by mutation of the C-terminal Gly residue of

the Ub moiety (UbG76VGagSL) resulted in more than threefold enhancement of SL-

presentation (Fig. 5.8 A). Alternatively to introduction of the SL epitope within the p2

spacer (GagSLp2), SL was added to the C-terminus of Gag variants (GagSLC-term.). In

addition, the sequence MSIINFEKL was introduced N-terminally of the wt protein

(GagSLN-term.). Analysis of SL-presentation derived from the N-terminus was not possible

Results 39

for Ub fusions due to the cotranslational cleavage of the Ub moiety. Comparison of SL-

presentation between the different constructs by double staining with 25D1.16-647 and

KC57-FITC as described above, clearly showed that only the internal position of SL

resulted in efficient MHC-I presentation (Fig. 5.8 A). While SL located at the C-terminus

of Gag was presented poorly by H2-Kb molecules, N-terminal SL seemed to be

completely destroyed by Ag processing and was not presented above background level

that was determined by staining of cells expressing Gag lacking the SL epitope (Fig.

5.8 A).

In parallel, expression of the GagSL proteins and release of VLPs was analysed by

Western blotting using an anti-p24-specific Ab (Fig. 5.8 B). While Ub was efficiently

cleaved from UbMGagSL and UbRGagSL fusions, UbG76VGagSL migrated as a stable

fusion protein of about 100 kDa (Fig. 5 8 B). As a loading control, blots were stripped

and reprobed using an anti-β-actin Ab. Although introduction of the SL epitope alone did

not affect the release of VLPs (compare lanes 1 and 2 in Fig. 5.8 B), expression as an Ub

fusion protein clearly interfered with budding. While the UbM fusion, which corresponds

to wt Gag after Ub cleavage, is only mildly attenuated in terms of budding, release of

VLPs is severely impaired by exchange of the N-terminal aa for Arg, both in the context

of UbRGagSLp2 (Fig. 5.8 B, lane 4) and UbRGagSLC-term (Fig. 5.8 B, lane 9), and almost

completely abolished by stable fusion to Ub (Fig. 5.8 B, lane 5).

Taken together, targeting of HIV-1 Gag for degradation by the UFD pathway

tremendously enhances MHC-I presentation of the Gag-derived SL epitope in HeLa-Kb

cells. Targeting Gag for the N-end rule pathway has similar, but less pronounced effects

compared to those observed in murine EL4 cell lines. Moreover, these results point out

that the kinetics of Ub removal might be different between murine EL4 cells and human

HeLa-Kb cells, as the UbMGagSL variant behaves different from the wt protein in terms

of MHC-I Ag presentation and VLP-release.

5.1.9 N-end rule and UFD degradation signals do not influence the synthesis or

metabolic half-life of Gag in HeLa cells

It was hypothesized that an increased turn-over of Gag might slow the rate of

accumulation in cell when transiently expressed and, finally, lead to a diminished steady-

state level. Although it was observed that all mutants reached similar steady-state protein

levels after transfection of Gag-encoding expression plasmids in a human HeLa-derived

cell line, we performed kinetic analyses to test if the mutants were synthesized at

Results 40

comparable rates, given that syngag is expressed very rapidly and efficiently and, thus,

saturation effects may occur in human cells. Therefore, HeLa cells were transfected with

individual syngag expression plasmids and cell samples as well as cell culture

supernatants were collected at different time points after transfection. Gag was detected

by anti-p6 Western blotting following SDS-PAGE of cell lysates and VLPs. Being

detectable as soon as four hours after transfection, increasing amounts of Pr55

accumulated with comparable kinetics, yielding stable protein levels approximately

12 hours after transfection (Fig. 5.9 A).

Fig. 5.9: N-end rule or UFD degradation signals do not influence the synthesis or the metabolic half-life of Gag. (A) HeLa cells were harvested at indicated points after transfection of syngag expression constructs and Gag synthesis was monitored by Western blotting using anti-p6 serum. Blots are representative of three independently performed experiments. (B) For pulse-chase experiments, HeLa cells were transfected with syngag expression constructs, radiolabelled for 20 min using [35S]Met and chased for up to 48 h. Pr55 was recovered from cell lysates by IP using anti-p6 and anti-p24 Abs, followed by SDS-PAGE and fluorography. (C) Densitometric quantification of [35S]-labeled Pr55. The radioactivity of the Pr55 band was quantified using a phosphor imager and plotted as percentage of the initial signal. Values represent mean +/- SD of three independent experiments.

Given the clear differences between the Gag variants in terms of MHC-I Ag processing

(see Fig. 5.4 and Fig. 5.8), it was reasonable to assess the metabolic half-life of Gag by

pulse-chase experiments. Following metabolic labeling of HeLa cells transiently

transfected with Gag expression plasmids with [35S]Met, Pr55 was recovered from cell

lysates by IP using a mixture of Gag-specific Abs. As shown in Fig. 5.9 B+C, all Gag

Results 41

variants displayed a metabolic half-life of approximately 9 hours comparable to the wt

protein. In summary, none of the degron signals tested in this study significantly affected

the stability of Pr55.

5.1.10 N-end rule and UFD degradation signals interfere with the release of VLPs

Although none of the degradation signals markedly affected the half-life or steady-state

level of Gag, they could still impair its correct folding during or shortly after synthesis

and, thus, enhance the DRiP-rate of Gag, as shown in EL4 cells (Fig. 5.3). Accumulation

of Gag-DRiPs can interfere with the highly ordered processes of assembly, budding and

release of VLPs. Steady-state Western blot analysis (see Fig. 5.8) already indicated a

budding defect of UbRGagSL and UbG76VGagSL. Therefore, the efficiencies of VLP

release, being regarded as indicative of the functionality of the Gag protein, were more

precisely quantified by time course Western blot analyses. Equal numbers of HeLa cells

were aliquoted 24 hours posttransfection after extensive washing in ice-cold PBS. VLPs

released into the cell culture medium during an incubation period of up to four hours were

pelleted through a sucrose cushion. Cell- and VLP-associated Gag was analyzed by SDS-

PAGE followed by immunoblotting with anti-p6 antiserum (Fig. 5.10 A). Membranes

were reprobed for β-actin as a loading control.

Following densitometric evaluation of Pr55-specific bands, release efficiencies were

calculated as [Pr55 (VLPs)*100 %/ Pr55 (VLPs + Cell)] and budding of the Gag variants

was compared (Fig. 5.10 B, n=3). Although it should correspond to wt Gag after cleavage

of the Ub moiety, the cleavable Ub fusion to the N-terminus (UbMGag), exhibited a

release efficiency reduced by half when compared to the wt protein (Fig. 5.10 B).

However, when the N-terminal Met was replaced by an Arg residue (UbRGag) or when

Ub remained attached to Gag at the N-terminus (UbG76VGag), the amount of released

VLPs dropped to almost undetectable levels, similar to those observed after expression of

a myristoylation-deficient Gag mutant (GagG2A; data not shown).

The Western blot results were confirmed by thin-section electron microscopy

(Fig. 5.10 C). HeLa cells were transiently transfected with psyngag and variants thereof

and, 24 h posttransfection, cultivated for further 24 h in cellulose capillary tubes to

prevent free diffusion of released virions, thus avoiding the need for centrifugation that

could affect VLP structure. Untransfected cells served as negative control (mock). In

contrast to HeLa cells expressing wt Gag or UbMGag, from which numerous VLPs with

typical immature morphology were released, only few viral structures were detected

extracellularly after expression of UbRGag. Expression of UbG76VGag resulted in the

Results 42

formation of enlarged and deformed extracellular structures that could not be

unambiguously identified as VLPs.

Fig. 5.10: N-end rule and UFD degradation signals interfere with the release of VLPs derived from synGag. (A) Time-course Western blot analysis of VLP release. Equal numbers of HeLa cells that were transiently transfected with Gag expression plasmids were aliquoted, cultured for up to 4 h while shaking and collected at the indicated time points. VLPs pelleted from the cell culture supernatant through a 20 % sucrose cushion and cell lysates were subjected in parallel to SDS-PAGE and Western blotting using an anti-p6 antiserum. (B) Densitometric quantification of VLP release. Gag-specific bands in Western blots from three independent experiments were quantified using the software AIDA. VLP release calculated as the Gag fraction contained in pelleted VLPs relative to total Gag was plotted against time. Mean values +/- SD are shown. (C) Electron microscopic analysis of VLP release. Transmission EM pictures of the PM of HeLa transfected with Gag expression plasmids.

43

5.1.11 N-end rule and UFD degradation signals disturb the membrane localization

of Gag

For targeting of HIV-1 Gag to membranes and thus, for the assembly, budding and

release of virus or VLPs at the PM, myristoylation at the Gly residue at position 2 is

essential. Hence, the inability of UbG76VGag to form VLPs could be easily explained by

the absence of an N-terminal myristoylation site in the protein. However, the negative

impact of cleavable N-terminal Ub fusion on budding and release of synGag-derived

VLPs could be caused by a defect in myristoylation and localization. Based on the

observation, that proteasome inhibition, leading to the accumulation of DRiPs, interferes

with HIV-1 budding and maturation (90), it has been proposed that Gag DRiPs might

disturb budding by preventing the ordered assembly of functional Gag molecules in a

prion-like manner (89, 91, 123).

To test whether Ub fusions to the N-terminus of Gag disturb the localization,

immunocytochemistry as well as subcellular fractionation was performed using HeLa

cells transiently transfected with psyngag expression plasmids.

Direct immunofluorescence staining using a FITC-conjugated anti-p24 Ab (KC57-FITC)

revealed that, in agreement with the data obtained in EL4 cells (136), Gag wt and

UbMGag are diffusely distributed in the cytoplasm and at the PM of transfected HeLa

cells (Fig. 5.11 A and B). In a fraction of all cells, a more intense perinuclear staining was

detected. UbRGag and UbG76VGag, however, show a more punctuate staining pattern that

could be the result of Gag protein aggregates. Thus, the exchange of the N-terminal Met

residue for Arg affects the subcellular distribution of Gag, though not dramatically.

Similar results were obtained when GagSL-GFP fusion proteins were visualized instead

of Gag immunostaining following expression of syngag constructs (data not shown).

To further substantiate this notion and to clarify if Gag can still bind to membranes,

membrane flotation by density gradient centrifugation was performed. Therefore, cells

were homogenized by sonification and nuclei, as well as unbroken cells were removed by

centrifugation at 2000 x g. The cleared homogenate was subjected to membrane flotation

on an Optiprep gradient and, following centrifugation for 5 h at 150,000 x g, eight

fractions were collected from the top of the gradient, denatured and examined by SDS-

PAGE and Western blotting using anti-p6 Ab. To control the fractionation, the

distribution of cellular proteins localized in the membrane fraction (Transferrin receptor,

TfR) or soluble fraction (ribosomal P antigen, RP0) was assessed using specific Abs.

Though TfR was also found in the two bottom fractions, possibly due to solubilization,

the RP0 was exclusively localized in the soluble bottom fractions.

44

Fig. 5.11: Analysis of Gag subcellular localization. (A-D) HeLa cells expressing Gag wt (A) or variants UbMGag (B), UbRGag (C) or UbG76VGag (D) were stained with the p24-specific KC57-FITC Ab. Nuclei were stained with DAPI. (E) Membrane flotation by density gradient centrifugation on an Optiprep gradient. HeLa cells expressing Gag variants were homogenized after removal of nuclei and unbroken cells and subjected to membrane flotation on a discontinuous Optiprep gradient. Fractions were collected from the top of the gradient, denatured and examined by SDS-PAGE and Western blotting using anti-p6 Ab. As a control, the distribution of the Transferrin receptor (TfR) and the ribosomal P Ag (RP0) was detected using specific Abs. (F) The fraction of membrane-associated Gag was calculated following densitometric quantification of anti-Gag Western blots obtained from three independent experiments. Mean values +/- SD are depicted.

Results 45

As a control, the unmyristoylated GagG2A mutant, which is known to be unable to bind to

membranes, was analysed in parallel and detected only in soluble fraction (Fig. 5.11 B),

while wt Gag was readily found in membrane fraction. Having thus confirmed that

fractionation in these gradients depends on membrane localization of Gag, membrane

association of the Gag Ub-fusion variants was analysed. UbG76VGag, like GagG2A and as

expected, was entirely localized in the soluble fraction. Following densitometric

quantification of Gag signals in Western blots, the relative membrane association of the

Gag variants was calculated based on data from three independent experiments

(Fig. 5.11 C). Whereas approximately 55 % of wt Gag and 40 % of UbMGag was

localized in the membrane fraction, membrane association of UbRGag was significantly

reduced to background level that was defined by GagG2A and UbG76VGag.

Taken together, these data indicate that UbG76VGag as well UbRGag fail to associate with

cellular membranes which explains the budding defect observed for these variants. In

agreement with an only mildly attenuated release of VLPs, UbMGag is still able to bind

to membranes, though slightly less efficient when compared to the wt protein. As the

tendency of Gag to bind to membranes can be regarded as indirect evidence for its

myristoylation, these results underline that processing of the N-terminal Met residue

proceeding the acceptor Gly residue is a prerequisite for efficient myristoylation (242).

Results 46

5.2 The PTAP Late domain regulates ubiquitination and MHC-I

antigen presentation of HIV-1 Gag

5.2.1 The PTAP L-domain in the p6 region regulates budding of GagSL-derived

VLPs.

As the PTAP L-domain of HIV-1 Gag has been shown by others to affect the level of Gag

ubiquitination (114, 120, 121), we set out to test whether, besides its function in virus

release, the PTAP motif can also regulate the MHC-I Ag processing of Gag. The PTAP

motifs within the p6 region GagSL were exchanged for the sequence AIVA by site-

directed mutagenesis, resulting in mutants that lack one (ΔPTAP1) or both PTAP L-

domains (ΔPTAP2) as schematically depicted in Fig. 5.12 A.

Fig. 5.12 The PTAP L-domain in the p6 region regulates budding of GagSL-derived VLPs. (A) Schematic representation of syngag-encoded wt and PTAP-deficient GagSL proteins used in this study. (B) Impaired release of PTAP-deficient GagSL as quantified by Western blot time course analyses. (C) Densitometric evaluation of the blots shown in (B). The time course of VLP release was calculated as the percentage of Gag present in the VLP fraction relative to the total amount of Gag.

To test whether the PTAP L-domains are functional in terms of driving the release of

VLPs derived from unprocessed GagSL, Western blot time-course analyses of budding

were performed. Compared to wt GagSL, VLP-release was reduced by approximately

20 % when only one PTAP motif was mutated, and further reduced down to 60 % in the

absence of both PTAP L-domains (Fig. 5.12 B and C). This result supports the

functionality of the PTAP L-domain in the context of unprocessed Gag harboring the SL

epitope. In further experiments, the ΔPTAP2 mutant is analyzed and will be referred to as

ΔPTAP or the PTAP-mutant from hereon.

Results 47

5.2.2 The PTAP L-domain regulates ubiquitination of GagSL

Next, we wanted to test whether the PTAP L-domains can regulate the ubiquitination of

syngag-encoded, unprocessed GagSL. A quick cell lysis was performed under conditions

where the proteasome and DUBs were inhibited, and the whole cell lysates were

subjected to SDS-PAGE and Western blotting using Gag-specific Abs. A ladder of bands

reminiscent of ubiquitinated Gag species migrating slower than Pr55 was detected when

the PTAP-mutant was expressed. These bands were virtually absent in case of the wt

GagSL protein (Fig. 5.13 A).

Fig. 5.13 The PTAP L-domain regulates ubiquitination of GagSL. (A) Western blot (WB) of whole HeLa cell lysates expressing wt or PTAP-deficient GagSL. As a loading control, the blot was reprobed using an anti-β-actin antibody. (B) IP of ubiquitinated Gag species. Following coexpression of GagSL with HA-tagged Ub in HeLa cells, Gag was precipitated under denaturing conditions from cell lysates using anti-HIV serum prebound to protein G-sepharose. Ubiquitinated Gag was detected by anti-HA Western blotting. The membrane was stripped and reprobed with anti-HIV to demonstrate that equal amounts of Gag protein were precipitated (lower panel). (C) Reduced recovery of polyubiquitinated Gag species after coexpression of HA-UbK48R mutant. After coexpression of wt Gag or the PTAP-mutant with HA-Ub, HA-UbK48R or HA-UbK63R, Gag was immunoprecipitated and ubiquitinated species were detected by anti-HA Western blotting. The membrane was reprobed with anti-HIV (lower panels).

To confirm that mutation of PTAP augments ubiquitination of GagSL, N-terminally HA-

tagged Ub (HA-Ub) was coexpressed with GagSL variants in HeLa cells. GagSL was

immunoprecipitated from denatured cell lysates using a pooled serum from HIV-1

infected patients. The precipitates were subjected to SDS-PAGE, and GagSL-HA-Ub

conjugates were visualized by anti-HA Western blotting. This allowed for the specific

Results 48

detection of ubiquitinated Gag, as the signal is absent when either GagSL or HA-Ub were

expressed alone (Fig 5.13 B, lanes 1, 2 and 5). GagSL-HA-Ub conjugates were detected

as two discrete bands and a smear characteristic of polyubiquitinated protein species.

Although similar quantities of GagSL were precipitated (Fig 5.13 B, lower panels), higher

amounts of ubiquitinated GagSL were recovered after mutation of the PTAP motifs

(Fig 5.13B, upper panels).

To investigate the type of Ub linkage, GagSL wt or ΔPTAP were coexpressed with

mutant forms of HA-Ub, carrying single Lys to Arg substitutions at either Lys48 (HA-

UbK48R) or Lys63 (HA-UbK63R). Overexpression of these Ub mutants has been shown to

interfere with the formation of Lys48- or Lys63-linked poly-Ub chains, respectively (243,

244). Due to the abundance of wt Ub in mammalian cells (245), HeLa cells were first

transfected with expression plasmids coding for Ub mutants and, 48 hours later, with Ub

expression plasmids and GagSL-GFP expression plasmids together. When the PTAP-

mutant was coexpressed HA-UbK48R, the intensity of the signal was reduced to wt levels

(Fig. 5.13 C). Overexpression of the HA-UbK63R mutant resulted only in slightly reduced

recovery of ubiquitinated GagSLΔPTAP (Fig. 5.13 C). These results indicate that

mutation of the PTAP L-domains results in predominantly Lys48-linked

polyubiquitination of GagSL.

5.2.3 The PTAP, but not the YP(X)nL L-domain regulates MHC-I antigen

presentation of a Gag-derived epitope

To investigate whether the increased ubiquitination of the PTAP-mutant correlates with

enhanced class-I presentation of Gag-derived SL, HeLa-Kb cells were transfected with

expression plasmids that code for GagSL-GFP fusion proteins. Flow cytometry using the

mAb 25D1.16 revealed that cells expressing PTAP-deficient Gag displayed

approximately two- to threefold higher numbers of H2-Kb-SL complexes at the cell

surface when compared to cells expressing the wt protein (Fig. 5.14 A). Indirect effects

on the MHC-I pathway causing variations in the total amount of cell surface MHC-I

could be excluded by staining with a mAb that specifically binds to H2-Kb molecules

irrespectively of the epitope that is displayed (Fig. 5.14 B).

Evidently, the amount of protein available for Ag processing intracellularly is one

determining factor for the number of epitopes derived from that Ag presented on the cell

surface (246).

Results 49

Fig. 5.14 The PTAP, but not the YP(X)nL L-domain, regulates MHC-I antigen presentation of a Gag-derived epitope. (A) Following transfection of HeLa-Kb cells with expression plasmids coding for GagSL-GFP wt or ΔPTAP, H2-Kb-SL complexes presented on the surface of GFP-positive cells were quantified by flow cytometry using the mAb 25D1.16 and a secondary Alexa647-conjugated anti-mouse Ab. To assess unspecific binding of the secondary Ab, cells were stained with secondary Ab only (control). A representative histogram plot is shown. (B) Samples were stained in parallel with cell culture supernatant of the hybridoma cell line B8-24-3 (B8), followed by staining with secondary Alexa647-conjugated anti-mouse Ab to assess total cell surface H2-Kb. (C) Quantification of SL-presentation including a GagSL-GFP variant with a mutated YP(X)nL motif (ΔYP). The mean fluorescence intensity (MFI) of the 25D1.16 staining was normalized to GFP fluorescence. Bars represent mean values +/- SD. (n=7; ΔYP n=3; * = P<0.05; ** = P<0.01). (D) More efficient T-cell activation by PTAP-deficient GagSL in vitro. Activation of B3Z hybridoma T cells was assessed by a colorimetric β- Gal assay after overnight cocultivation with HeLa-Kb cells expressing GagSL wt or ΔPTAP in various effector to target ratios. (E) Western blot analysis of GagSL variants. HeLa cells expressing wt GagSL or variants ΔPTAP or ΔYP were lysed and whole cell lysates as well as VLPs pelleted from the cell culture supernatant were subjected to SDS-PAGE and Western blotting using anti-p24 antiserum.

Results 50

In order to compensate for possible differences in expression levels of the GagSL-GFP

variants, we normalized the MFI of the staining with 25D1.16 to the MFI of the GFP

fluorescence in seven independent experiments, revealing a significant increase in SL-

presentation when the ΔPTAP variant was expressed when compared to the wt

(Fig. 5.14 C).

To exclude that the augmentation of SL-presentation associated with disruption of the

PTAP motif results from introduction of the motif AIVA, the PTAP L-domain was

alternatively exchanged for the sequence LIRL, which is commonly used in full-length

HIV-1 expression systems to avoid substitutions in the overlapping pol reading frame.

Intriguingly, the increase in the number of H2-Kb-SL complexes presented on the cell

surface of cells expressing a PTAP-mutant was almost identical, regardless of whether

PTAP was exchanged for AIVA or LIRL (data not shown).

In addition to the primary PTAP L-domain, an auxiliary YP(X)nL L-domain motif is

located more C-terminally within p6. To answer the question whether the second L-

domain in HIV-1 Gag is also involved in the regulation of MHC-I Ag processing, the

sequence YPLTSL was exchanged for RSLTSL in the context of GagSL, and SL-

presentation of this ΔYP-mutant was analyzed. Staining with 25D1.16, as described

above, revealed that the number of H2-Kb-SL complexes displayed at the surface of cells

expressing the ΔYP mutant is identical to those expressing wt GagSL (Fig. 5.14 C). To

assess the release of VLPs derived from the ΔYP mutant, VLPs were pelleted from the

cell culture supernatant of HeLa cells transfected with expression plasmids coding for

GagSL wt or the variants ΔPTAP or ΔYP, respectively. Consistent with the kinetic data

(Fig. 5.12 B), only minor amounts of VLPs were detected following expression of

GagSLΔPTAP, whereas the ΔYP mutant was comparable to the wt protein with respect to

VLP release (Fig. 5.14 D). In Western blot analyses of whole cell lysates using a Gag-

specific Ab, GagSLΔPTAP displayed a ladder of ubiquitinated Pr55 (see also

Fig. 5.13 A), while the ΔYP mutant shows no increased ubiquitination when compared to

the wt (Fig. 5.14 D).

To confirm the results obtained by flow cytometry, we tested the ability of HeLa-Kb cells

expressing GagSL either in its wt form or with a mutated PTAP motif, to activate B3Z

hybridoma T cells. As a negative control, HeLa-Kb cells were transfected with an empty

vector (mock). After overnight cocultivation, β-Gal activity was measured by a

colorimetric assay, showing that cells expressing the PTAP-mutant more efficiently

activate T cells over a broad range of effector-to-target ratios when compared to cells

Results 51

expressing the wt protein (Fig. 5.14 E). Taken together, these data indicate that the PTAP

L-domain can indeed regulate the entry of Gag into the MHC-I pathway.

5.2.4 Induction of the immunoproteasome enhances presentation of the SL-epitope

derived from GagSL-GFP

In vivo, naïve CD8+ T cells are predominantly primed by DCs, which express not only the

constitutive subunits of the 26S proteasome, but also the immunosubunits β1i (LMP2),

β5i (LMP7), β2i (MECL1) and the 11S activator PA28 (40). The immunosubunits can be

induced by IFN-γ and replace the constitutive subunits in nascent proteasomes to build

the immunoproteasome (37, 38). Immunoproteasomes have been described to have

altered cleavage specificities, resulting in a modified spectrum of class I-presented

epitopes (41), and an enhanced cleavage capacity (43-47). Recently, IFN-γ has been

shown to induce oxidative stress in cells, leading to the accumulation of DRiPs that are

subsequently cleared by the enhanced proteolytic activity of newly formed

immunoproteasomes (247). This finding points towards a more general role for IFN-γ in

the generation class-I peptide ligands.

Fig. 5.15: Induction of the immunoproteasome enhances presen-tation of the SL-epitope derived from GagSL-GFP wt and ΔPTAP. (A) HeLa-Kb cells expressing GagSL-GFP wt or ΔPTAP were treated with 250 U of IFN-γ for 16 h or left untreated as a control. H2-Kb-SL complexes presented on the surface of GFP-positive cells were quantified by flow cytometry using the mAb 25D1.16 conjugated to APC. The MFI of the 25D1.16 staining was normalized to the MFI of the GFP fluorescence. Bars represent mean values +/- SD from three independent experiments. (B) Aliquots of the cells used in (A) were lysed, subjected to SDS-PAGE and Western blotting. GagSL-GFP was detected using a pooled serum from HIV-1-positive patients. Induction of the immunoproteasome was confirmed by staining against the inducible subunit β5i. The blot was stripped and reprobed using an antiserum directed against the constitutive proteasome subunit β5. Staining for β-actin served as a loading control.

Results 52

Therefore, it was tested whether induction of immunoproteasomes affects the Ag

processing of GagSL. HeLa-Kb cells expressing GagSL-GFP wt or ΔPTAP were treated

with IFN-γ for 16 h and SL-presentation was assessed using fluorescently labeled

25D1.16 Ab. Cells expressing GFP served as negative control. IFN-γ treatment resulted

in augmentation of MHC-I presentation of the SL-epitope derived from GagSL wt and

ΔPTAP (Fig. 5.15A). An increased level of β5i (LMP7) as detected by Western blotting

(Fig. 5.15B). served as control for induction of immunoproteasomes by IFN-γ. This result

indicates that first, IFN-γ has an overall positive effect on the presentation of the SL

epitope derived from GagSL and second, that Gag-DRiPs are not only a bona fide

substrate for constitutive standard proteasomes (133), but also for IFN-γ induced

immunoproteasomes.

5.2.5 The PTAP L-domain regulates MHC-I antigen presentation of the SL epitope

derived from processed Gag

We next asked whether the PTAP L-domain not only regulates entry of the unprocessed

Pr55 polyprotein precursor, but also of processed Gag into the MHC-I pathway. To

answer this question, the SL-coding sequence has been introduced into the proviral

constructs pNL4-3 (248), pBRNL4-3nef-IRES-GFP, carrying an internal ribosome entry

site (IRES) followed by the gfp gene (249), and the HIV-1NL4-3 subgenomic expression

vector pNLenv, in which the env gene was deleted (250). Thereby, the SL epitope is

located in the p2 spacer region corresponding to its position in syngag-encoded Pr55. To

prevent alterations in the overlapping pol-reading frame, the PTAP-motif was exchanged

for the sequence LIRL.

Following transfection of HeLa-Kb cells with pBRNL4-3nef-IRES-GFP coding for

GagSL or a PTAP-mutant, H2-Kb-SL complexes presented on the surface of GFP-

positive cells were analysed by flow cytometry using the mAb 25D1.16. Cells expressing

pBRNL4-3nef-IRES-GFP encoding Gag wt or ΔPTAP, but lacking the SL epitope served

as a negative control for SL-presentation (data not shown). In contrast to the experiments

using syngag-encoded Pr55, cells expressing wt GagSL from a full-length proviral

construct displayed only few H2-Kb-SL complexes at the cell surface, as evident from a

weak specific staining with 25D1.16, whereas mutation of the PTAP L-domain

dramatically increased SL-presentation (data not shown).

Results 53

Fig. 5.16 The PTAP L-domain regulates MHC-I presentation of the SL-epitope introduced into full-length HIV-1. (A) Following transfection of HeLa-Kb cells with proviral constructs pBRNL4-3nef-IRES-GFP coding for wt Gag or a PTAP-mutant, both harboring the SL-epitope within the p2 spacer region, H2-Kb-SL complexes presented on the surface of GFP-positive cells were quantified by flow cytometry using the mAb 25D1.16. The MFI of the 25D1.16 staining was normalized to GFP

Results 54

fluorescence. Bars represent mean values +/- SD (n=4; * = P<0.05). (B) HeLa-Kb cells were transfected with pNLenv encoding Gag wt or ΔPTAP followed by extracellular staining using 25D1.16-PE and intracellular staining using a FITC-labeled p24-specific mAb (KC57-FITC). Bars represent mean values +/- SD after normalization to the amount of intracellular Gag (n=7; ** = P<0.01). (C) Western blot analysis of Gag proteins. Cell lysates of HeLa cells transfected with pNL4-3 proviral or pNLenv constructs coding for Gag wt or a PTAP-mutant harboring or lacking the SL-sequence, respectively, were subjected to Western blotting using anti-p24 antiserum. Membranes were reprobed with an anti-β-actin antibody as a loading control. Virions contained in the cell culture supernatant were pelleted through 20 % (w/v) sucrose, lysed, and p24 was detected by Western blotting. (D) Release of infectious units from transfected cells was analysed by quantification of β-Gal activity after infection of HeLa-TZM-bl reporter cells.

The MFI of the 25D1.16 staining was normalized to GFP fluorescence and quantification

of three independent experiments clearly revealed higher numbers of H2-Kb-SL

complexes on cells expressing PTAP-deficient Gag (Fig. 5.16 A; ΔPTAP-SL) when

compared to cells expressing wt Gag (Fig. 5.16 A; wt-SL). The increase in SL-

presentation in absence of the PTAP-motif is higher when Gag was expressed in context

of full-length HIV-1 when compared to expression of Gag alone (compare Fig. 5.14 and

Fig. 5.16 A). To exclude that differences in the numbers of H2-Kb-SL complexes were

caused by variations in the abundance of H2-Kb molecules on the cell surface, cells were

stained in parallel with a H2-Kb-specific Ab, revealing equal numbers of these MHC-I

molecules on the surface of all HIV-1 expressing cells (data not shown).

Similar experiments that were conducted using the expression plasmid pNLenv coding

either for wt or PTAP-deficient GagSL (Fig. 5.16 B) confirmed that the PTAP L-domain

not only regulates MHC-I Ag presentation of the SL epitope derived from unprocessed

Pr55, but also of Gag that undergoes processing by the viral PR.

In order to characterize the HIV-1 variants harboring the SL epitope within the p2 spacer

of Gag with respect to Gag processing and virus release, HeLa cells were transfected with

pNL4-3 or pNLenv and variants thereof, followed by Western blot analysis of cell lysates

and virus pelleted from the cell culture supernatant using Gag- or HIV-1-specific

antibodies. For PTAP-deficient HIVNL4-3, the characteristic defect in Gag processing with

accumulation of intermediate processing products as well as reduced virus release was

observed (Fig. 5.16 C, lanes 2, 4, 6, 8). Independently of the presence of the SL-epitope,

also a ladder of ubiquitinated Pr55 was detected, as already observed for syngag-encoded

GagSL. We found that introduction of the SL-sequence into the p2 spacer resulted in an

altered processing of p24/p25. When SL was introduced in context of wt Gag, the p25

band became virtually undetectable (Fig. 5.16 C, lanes 3 and 7). Introduction of SL in

context of the PTAP-mutant, however, produced two Gag species that could be detected

as a double-band (Fig. 5.16 C, lanes 4 and 8). In addition, a third, slower migrating band

Results 55

could be detected, representing CA fused to p2 containing the SL sequence. This

suggests, that introduction of the sequence SIINFEKL into the p2 spacer results either in

an altered processing of p25/p24 or affects the migration behavior of the p2-containing

Gag products.

In agreement with the processing defect, introduction of the SL-epitope reduced the

specific infectivity of the virus by approximately 3-fold as determined by single round

infection of HeLa TZM-bl reporter cells with equal amounts of virus as quantified by p24

antigen ELISA (Fig. 5.16 D). These cells harbor the lacZ and luciferase reporter genes

under the control of the HIV-1 long terminal repeat (LTR) promoter. As expected,

mutation of PTAP resulted in a loss of infectivity, however, even PTAP-deficient virus is

further impaired with respect to infectivity in the presence of SL in the p2 spacer.

Replication studies in Jurkat T cells (data not shown, performed by Christian Setz)

showed an impaired replication capacity of SL-containing viruses, further substantiating

the decreased infectivity observed in single-round assays. These results point out that the

length and sequence of the p2 spacer peptide might play an important role for Gag

processing and virus infectivity.

5.2.6 Enhanced SL-presentation of the PTAP-mutant is not a result of the budding

defect and not entirely dependent on membrane association of Gag

The enhanced SL-presentation observed after expression of a GagSL-GFPΔPTAP

(Fig. 5.14) could be easily explained by an increased amount of Gag protein available for

Ag processing within the cell due to the budding defect of the PTAP-mutant (Fig 5.12 B,

5.14 D). To test this hypothesis, we used a Gag mutant with a single aa substitution at

position 2 (G2A), which can not be myristoylated. As the cotranslational attachment of a

myristate group to the N-terminal Gly residue is essential for the association of Pr55 with

membranes (4, 6), this G2A mutant is, like the PTAP-mutant, incapable of budding. If the

induction of a budding defect were sufficient to enhance Ag presentation, this

myristoylation-deficient mutant should also display increased SL-presentation.

Nevertheless, the number of SL-H2-Kb complexes at the surface of cells expressing the

G2A-mutant did not exceed the amount displayed on cells expressing the wt protein

(Fig. 5.17 A), although VLP-release was completely abolished (Fig. 5.17 B). Mutation of

the myristoylation site in context of the PTAP-mutant, however, resulted in increased

class I presentation of the Gag-derived SL epitope, indicating that the enhanced SL-

Results 56

presentation is not caused by the budding defect and not entirely dependent on membrane

association of Gag, but might be specifically regulated by the PTAP motif.

Fig. 5.17: Enhanced MHC-I antigen presentation of the PTAP-mutant is not a result of the budding defect and not entirely dependent on membrane association of Gag. (A) HeLa-Kb cells were transfected with plasmids encoding GagSL-GFP wt or ΔPTAP and the number of H2-Kb-SL complexes on the surface of GFP-positive cells was quantified by flow cytometry. The MFI of the 25D1.16 staining was normalized to GFP fluorescence. Mean values +/- SD are shown (n=4; * = P<0.05). (B) Release of VLPs derived from GagSL-GFP variants was quantified by anti-p24 immunoblotting.

5.2.7 The interaction with Tsg101 or ALIX is not essential for the regulation of

MHC-I presentation of a Gag-derived epitope by the PTAP L-domain

The PTAP motif has been shown to bind to Tsg101 (13), a component of the ESCRT-I,

and this interaction is essential for the recruitment of cellular factors to support HIV-1

budding (11, 12). The disruption of this interaction by mutation of the PTAP-motif might

influence the ubiquitination of Gag, possibly due to the hampered recruitment of some yet

unidentified DUB, and, thus, affect the entry of Gag into the MHC-I pathway. We

therefore asked whether the disturbance of the interaction between the PTAP L-domain

and Tsg101 by siRNA-mediated depletion of Tsg101 also leads to increased SL-

presentation as does mutation of PTAP. Although knockdown of Tsg101 with 77 %

efficiency could be achieved by transfection of HeLa-Kb cells with Tsg101-specific

siRNA (Fig. 5.18 B), this did not lead to increased SL-presentation after coexpression of

GagSL-GFP (Fig. 5.18 A).

Overexpression of ALIX, that interacts with the ESCRT-III via CHMP4B and binds to

the secondary YP(X)nL L-domain of HIV-1, can overcome the budding defect induced by

mutation of the primary PTAP L-domain (22). Therefore, we tested whether restoration of

the interaction with the ESCRT and rescue of the budding defect by ALIX overexpression

has any influence on the presentation of the SL epitope derived from PTAP-deficient

GagSL. GagSL-GFP wt or ΔPTAP were coexpressed with Flag-tagged ALIX in HeLa-Kb

cells and SL-presentation was quantified by staining with 25D1.16. Expression of ALIX

Results 57

was confirmed by Western blotting (Fig. 5.18 D). Although cotransfection of increasing

amounts of pFLAG-ALIX resulted in a rescue of VLP-release of PTAP-deficient GagSL-

GFP as quantified by Western blotting of cell lysates and VLPs (Fig. 5.18 D),

overexpression of ALIX did not affect the MHC-I presentation of the Gag-derived SL

epitope (Fig. 5.18 C).

Fig. 5.18: The interaction with Tsg101 or ALIX is not essential for the regulation of MHC-I presentation of a Gag-derived epitope by the PTAP L-domain. (A) Following transfection of HeLa-Kb cells with Tsg101-specific or a scrambled control siRNA and, 48 h later, with siRNA and expression plasmids coding for GagSL-GFP wt or ΔPTAP, SL-presentation was assessed by flow cytometry using the mAb 25D1.16. Bars represent mean +/- SD after normalization to GFP fluorescence (n=4; * = P<0.05). (B) Knockdown of Tsg101 was confirmed by Western blotting. (C) Quantification of mean SL-presentation normalized for GFP fluorescence after coexpression of GagSL-GFP wt or ΔPTAP and ALIX or an empty vector control (mock). Bars represent mean +/- SD (n=4; * = P<0.05). (D) Rescue of VLP-release by ALIX overexpression. HeLa-Kb cells were transfected with plasmids coding for GagSL-GFP wt or ΔPTAP and increasing amounts of pCMV-Flag-ALIX (0, 0.5 or 1 µg). VLP release was assessed by staining of cell and VLP fractions with anti-p24 Ab. Expression of ALIX was confirmed by anti-Flag Western blotting.

These data point out that the disruption of the interaction between the PTAP-motif

located within the p6 domain of HIV-1 Gag and the UEV domain of Tsg101, that

mediates recruitment of the ESCRT-I, does not represent a likely explanation for the

enhanced entry of Gag lacking a functional PTAP L-domain into the MHC-I pathway. In

agreement with the results obtained for the G2A mutant (Fig. 5.17), impairment of VLP-

release alone, induced here by knockdown of Tsg101, is not sufficient to increase SL-

presentation. Consistently, MHC-I presentation of the Gag-derived SL epitope is not

markedly influenced by the rescue of the budding defect caused by the PTAP-mutation by

overexpression of ALIX.

Results 58

5.2.8 Lys48-linked polyubiquitination is essential for the preferred entry of the

PTAP-mutant into the MHC-I pathway

Although Ub-independent access to the proteasome has been described (reviewed in

(49)), most substrates become tagged for proteasomal degradation by the attachment of at

least four residues of Lys48-linked Ub (251). To test whether polyubiquitination of Gag is

crucial for the better entry of the PTAP-mutant into the MHC-I pathway, GagSL-GFP

expression plasmids were coexpressed with expression plasmids coding for Ub mutants

UbK48R, UbK63R or UbK48,63R in HeLa-Kb cells. To obtain sufficiently high expression

levels of Ub mutants, a consecutive transfection protocol was used as described in section

5.2.2.

SL-presentation was assessed using staining with 25D1.16 as described above. While

overexpression of wt Ub, used as a control, had no effect on SL-presentation, interference

with Lys48-linked poly-Ub chain formation reduced the MHC-I presentation of SL

derived from both, Gag-SL-GFP wt and ΔPTAP2 to background levels (Fig. 5.19). In

agreement, a reduction of SL-presentation of the same magnitude was obtained after

cotransfection with the double mutant UbK48,63R, while expression of the single mutant

UbK63R had only marginal effects on SL-presentation. As massive overexpression of

UbK48R might interfere with cell viability in general, in particular with proteasome activity

or MHC-I Ag presentation, the number of total H2-Kb molecules at the cell surface were

monitored in parallel. However, no changes in total H2-Kb could be detected following

expression of any of the Ub variants (data not shown), which indicates that the overall

MHC-I Ag presentation pathway was still functional. These results point out that the

attachment of Lys48-linked poly-Ub chains, the canonical signal for proteasomal

degradation, regulates the entry of the PTAP-deficient Gag into the MHC-I Ag

presentation pathway.

Fig. 5.19: Lys48-linked polyubiquitination is essential for the preferred entry of the PTAP-mutant into the MHC-I pathway. Following coexpression of GagSL-GFP wt (black bars) or ΔPTAP (grey bars) with Ub or Ub mutants in HeLa-Kb cells, H2-Kb-SL complexes at the cell surface were quantified by flow cytometry using 25D1.16 and the MFI was normalized to GFP. Bars represent mean +/- SD from three independent experiments.

Results 59

5.2.9 The PTAP-mutant displays a slightly decreased metabolic half-life and an

increased DRiP-rate when compared to wt Gag

It has been shown here that formation of poly-Ub chains on ΔPTAP GagSL molecules is

essential for increased SL-presentation (Fig. 5.19) and that the PTAP-mutant is

ubiquitinated to higher levels when compared to wt GagSL (Fig. 5.13), suggesting that

GagSLΔPTAP is preferentially recognized and degraded by the UPS. This led to the

question if this enhanced ubiquitination correlates with a reduced half-life of the protein.

Therefore, standard pulse-chase analyses were conducted using transiently transfected

HeLa cells that were pulsed with [35S]Met for 15 min followed by 6 hours of chase. Gag

was recovered from cell lysates by IP using a mixture of Gag-specific antibodies,

subjected to SDS-PAGE and analyzed by fluorography (Fig. 5.20 A).

Radioactivity in dried gels corresponding to the Pr55 band was quantified using

phosphorimaging instrumentation and software. Data from four independent experiments

show that the PTAP-mutant displays a slightly decreased metabolic half-life when

compared to wt GagSL (Fig 5.20 B). To test whether this decreased stability is

consequent to a rapid cotranslational turn-over, short-term DRiP pulse-chase experiments

were performed according to previously established protocols (133, 135, 136).

HeLa cells were transiently transfected with GagSL expression plasmids and treated with

a combination of zLLL (228) and the highly specific PI lactacystin during the final

10 min of a 30 min starvation period, throughout a 3 min pulse with [35S]Met as well as a

60 min chase period. dimethyl sulfoxide (DMSO)-treated cells served as a solvent

control. The complete shut-down of proteasomal activity at the time of metabolic labeling

allows for the detection and quantification of newly synthesized proteins that would

otherwise undergo cotranslational degradation and therefore escape detection.

Gag proteins precipitated by specific antibodies were resolved by SDS-PAGE and

analyzed by fluorography (Fig. 5.20 C). The quantities of radioactivity corresponding

either to Pr55 or the total proteins migrating in the MW range of approximately 60 to

250 kDa were measured using a phosphorimager (Fig. 5.20 D). In the absence of

proteasome inhibitors, the decline of Gag in cells expressing the PTAP-mutant or the wt

protein was almost identical. After proteasome shutdown, there is a transient increase in

recovery of Pr55 in the wt situation. In absence of a PTAP-sequence, however, the

magnitude of this increase is clearly higher. The amount of Pr55 recovered from cell

lysates was increased by more than 50 % immediately after the pulse and attained a

maximum level of up to 80 % within 5 min (Fig. 5.20 D, left panel). After reaching a

Results 60

certain plateau, the recovery of Pr55 declines during the last 30 min of chase, most

probably due to processes like ubiquitination, proteolytic cleavage and membrane

association.

Fig. 5.20: The PTAP-mutant displays a slightly decreased metabolic half-life and an increased DRiP-rate when compared to wt Gag. (A+B) For long-term pulse-chase experiments, transiently transfected HeLa cells were radiolabelled for 15 min with [ 35S]Met and chased for up to 6 hours. (A) Fluorograph of GagSL proteins recovered by IP using anti-p6 and anti-p24 antibodies and separated by SDS-PAGE. (B) Densitometric quantification of [ 35S]-labeled Pr55 using a phosphorimager. PSL: Photostimulated luminescence. Values represent mean and SD of four independent experiments. (C+D) For short-term DRiP pulse-chase experiments, transiently transfected HeLa cells treated with 20 µM of zLLL/LC or DMSO as a solvent control each during the final 10 min of a 30 min starvation period were pulsed for 3 min with [ 35S]Met and chased for up to 60 min the presence or absence of zLLL/LC. (C) Fluorograph of GagSL recovered by IP and resolved by SDS-PAGE. (D) Densitometric quantification of [35S]-labeled Pr55 (left panel) and the high molecular weight (HMW) smear recovered with anti-Gag antibodies (right panel) of the fluorograph shown in C.

Results 61

The smear of proteins migration in the MW range of 60 kDa to 250 kDa recovered by

Gag-specific antibodies is thought to represent polyubiquitinated Gag species (133, 136).

Quantification of this HMW smear of proteins showed that the PTAP-mutant is

ubiquitinated to higher levels and these polyubiquitinated Gag species accumulate over

time (Fig. 5.20 C and 5.20 D, right panel). The accumulation of these polyubiquitinated

species after proteasome shutdown was taken into account to assess the DRiP-rate of Gag.

Though treatment of cells expressing wt GagSL with zLLL/LC resulted in a minor

enhancement of recovery of proteins in this MW range, the magnitude of increase was

unambiguously higher in cells expressing the PTAP-mutant, reaching about 100 % after

15 min of chase (Fig. 5.20 D, right panel).

Taken together, pulse-chase analyses revealed that, although the metabolic half-life of

PTAP-deficient GagSL is not considerably diminished, its DRiP-rate is clearly enhanced

when compared to wt GagSL. These data provide an explanation for the increased number

of H2-Kb-SL complexes presented at the surface of cells expressing the PTAP-mutant.

Nevertheless, the mechanism how the lack of a functional PTAP-motif is recognized by

the cellular protein quality control system remains to be answered.

Discussion

62

6 Discussion

In this study, the HIV-1 Gag polyprotein was chosen as a model Ag to more precisely

understand the relevance of erroneous protein synthesis for the generation of MHC-I-

presented peptides. To this end, the effect of degradation signals artificially fused to HIV-

1 Gag on its efficiency of biogenesis, metabolic half-life, DRiP-formation as well as

subcellular localization, and VLP-release has been investigated in this work. These

parameters of Gag protein function have been correlated to the efficiency of MHC-I

presentation of a Gag-derived SL model epitope. It was demonstrated that the exchange

of the N-terminal Met residue for Arg, a destabilizing aa according to the N-end rule,

directed Gag to the DRiP-pathway and resulted in enhanced MHC-I Ag presentation, as

well as a better CD8+ T-cell response, both, in vitro and in vivo. Moreover, the PTAP L-

domain located within the C-terminal p6 region of Pr55 was identified to be a naturally

occurring sequence motif that, besides its already well characterized and essential role in

virus release, also governs the DRiP formation and Ag presentation of HIV-1 Gag.

Regardless of all the efforts spent so far, there is still no protective or therapeutic

vaccination against HIV-1 available. Recent vaccine trials failed to confer protection

against HIV-1 infection, but may help to generate new hypotheses that can be followed in

future research (252-255). Key obstacles for the development of an effective HIV-1

vaccine include, amongst others, the huge sequence variation of HIV-1. Moreover, the

natural immune response to HIV-1 does not protect against superinfection (256-258).

Therefore, a better understanding of what constitutes a protective immune response

against HIV-1 and of the immune responses induced by vaccination is crucial. Based on

this knowledge, it may be possible to enhance the immunogenicity of vaccine Ags to

induce immune responses that can prevent or control HIV-1 infection.

Though it is now generally accepted that the induction of Abs that neutralize a broad

spectrum of HIV-1 isolates is required to block acquisition of HIV-1 infection (219, 259),

virus-specific CD8+ T cells display a key function in the immune control of virus spread

(260), especially during acute viremia (261). In order to evade specific CTL responses,

HIV-1 quickly mutates MHC-I-restricted epitopes, showing that those responses exert

selective pressure (260-265). Moreover, certain MHC-I alleles have been correlated with

viral load and disease progression (262, 266-268). Recently, a genome-wide association

study suggested the binding properties of MHC-I molecules to be the major genetic factor

for the control of HIV-1 infection (269). Therefore, vaccine strategies aimed at the

Discussion

63

generation of potent virus-specific CTL responses are now generally considered as a

therapeutic option to interfere with disease progression subsequent to HIV infection

In this study, HIV-1 Gag was chosen as a model protein to assess strategies to enhance

MHC-I Ag presentation, since it has been demonstrated by several studies that an

effective CTL response acting specifically against HIV-1 Gag, but not against other viral

Ags, can be correlated with a significant reduction in viral load in HIV-1 infected patients

(220, 270-272) or in SIV-1 infected Rhesus macaques (273, 274). Importantly, immune

escape mutations within Gag-derived epitopes may be associated with a significant loss of

viral fitness (275-277). The Gag protein can therefore be regarded as an promising

vaccine Ag, and approaches that aim at optimizing the immunogenicity of HIV-1 Gag

might be useful to elicit a broad and sustained cellular immune response.

In addition, Gag is exclusively present in the cytosolic compartment and thus, fully

accessible to the UPS and entry into the MHC-I Ag processing pathway. Furthermore,

principal biochemical procedures to measure the DRiP-rate of Gag have been established,

and Gag-DRiPs have already been detected (133). Therefore, HIV-1 Gag can be

considered as an interesting model Ag.

Although a number of Gag-derived MHC-I epitopes have been characterized, there is still

no specific Ab available to analyze the quantity of Gag-derived epitopes in complex with

MHC-I molecules at the cell surface. To circumvent this problem we found that insertion

of the standard model epitope SL into the polymorphic p2 spacer region of Gag (278)

resulted in efficient presentation of SL on the cell surface that did not interfere with

budding and release of synGag-derived VLPs (see Fig. 5.8). However, though virus-

release was not markedly affected when SL was introduced into the p2 spacer of Gag

encoded by HIV-1NL4-3, an altered migration behavior in SDS-PAGE and/or processing

pattern of CA was observed (Fig. 5.15). This led us to speculate that introduction of

additional eight amino acids into p2 might alter the processing of the CA-p2 protein

(p25). One possible explanation is the alternative usage of a cleavage site within p2,

which has been previously proposed between position Met-4 and Ser-5 of p2 (226, 279,

280). Alternatively, an additional site inserted by introduction of the SL sequence may be

recognized by the PR, leading to a shortened p25 product that migrates as a double band

with p24. As the C-terminal domain of CA has been shown to adopt an α-helical structure

that extends into p2 (225, 226, 281), the changed migration behavior described here could

also reflect structural alterations induced by the extension of the p2 linker peptide.

Though this observation is not highly relevant to answer questions addressed in this work,

it indicates that the length and sequence of the p2 spacer might be important for Gag

Discussion

64

processing. However, more careful examination would be necessary to clarify this

phenomenon and to provide further insight into the role of the p2 spacer peptide during

the viral life cycle.

We found that the position of insertion clearly affected the efficiency of SL-presentation

(Fig. 5.8). Only insertion at the internal position (p2) resulted in efficient SL-presentation.

When the SL sequence was inserted at the ultimate C-terminus of Gag, the epitope was

only poorly presented by H2-Kb molecules, while insertion at the N-terminus did not

result in MHC-I presentation of the epitope at all. The 26S proteasome has to produce the

correct C-termini of MHC-I-presented peptides, whereas N-terminally extended antigenic

precursors can be trimmed by other proteases (153, 282, 283). Accordingly, proteasomal

processing might not always generate the correct C-terminus of the SL-epitope.

MSIINFEKL located at the N-terminus of Gag could undergo N-terminal trimming, for

example by ERAAP in the ER, leading to the destruction of the epitope. Depending on

the surrounding amino acids, the SL epitope may also be destroyed by cuts made within

the SL sequence by the proteasome.

Several studies have established that targeting an Ag for rapid degradation by the 26S

proteasome represents an effective approach to enhance MHC-I Ag presentation (206,

207, 210-214) and induction of CTL responses (216, 284-288). Motifs of the protein

primary sequence that govern protein stability have been identified in cellular (171) and

viral proteins (e.g. the GA-stretch of Epstein-Barr virus (EBV) that stabilizes EBNA-1

(289), an N-end rule degron in HIV-1 Integrase (200), and newly identified degradation

signals within the C-terminal regions of alphavirus nsP3 (290), Influenza C virus p42

(291) and Hantavirus G1 (292).

Initially, it was attempted to target HIV-1 Gag for rapid proteasomal degradation by the

introduction of various degradation signals or potentially destabilizing aa exchanges (see

Fig. 5.1). However, none of these degradation signals conferred instability to Gag in

terms of overall metabolic half-life, neither in murine nor in human cell lines (Fig. 5.2,

Fig. 5.9 and data not shown). The validity of the N-end rule has been successfully

demonstrated for a number of Ags derived from viruses and bacteria (206, 207, 211, 213,

214, 217). Nevertheless, the N-end rule does not seem to apply ubiquitously for all

proteins tested so far (210, 293). Targeting to the UFD pathway reduced the half-life of

most model substrates (205, 287, 294, 295), but fusion of a single copy of Ub had

sometimes only modest effect on protein half-life (293, 294). Our observation that the

metabolic half-life of Gag is only marginally influenced by the introduction of N-end rule

or UFD degradation signals is in agreement with previous findings made by others (210).

Discussion

65

Even addition of a short Lys-rich leader sequence to a similar UbRGag fusion protein

resulted only in moderate destabilization of Gag (210).

It is shown here that targeting of Gag to the N-end rule pathway clearly increases its

DRiP-rate (Fig. 5.3). This discrepancy might be explained by the possibility that N-

terminal degradation signals may be buried in the context of completely folded Gag. In

contrast, the destabilizing Arg-residue at the N-terminus of the nascent protein should be

better accessible to cognate Ub ligases during protein translation. Moreover, its inherent

capacity to self-assemble might contribute to the resistance of Gag to the N-end rule and

UFD. The formation of Gag multimeric complexes during budding could obscure the

recognition of degradation signals by cellular proteins. Although we can merely speculate

about the underlying reasons for the variable impact of N-end rule and UFD degradation

signals on protein half-life, it seems reasonable that polypeptides tagged for destruction or

damaged proteins are removed as quickly as possibly, preferentially during synthesis.

The reduced Gag steady-state level in RGag expressing cells when compared to the M-

Gag expressing cells might also point towards a rapid co-translational degradation of a

certain proportion of total Gag. This should slightly reduce the steady-state level of RGag

that otherwise exhibits the same turn-over rate during the post-translational “second”

half-life. The importance of protein de novo synthesis (234, 236) for efficient MHC-I

presentation, also found here for Gag-derived SL (Fig. 5.5), underlines the well

established concept that DRiPs represent the main source for MHC-I-presented peptides

(132, 133). Viral DRiPs have been demonstrated for HIV-1 Gag (133), IAV

nucleoprotein (212, 296) and, very recently, for IAV neuraminidase (297) and EBV

EBNA-1 (298).

There is only limited knowledge about naturally occurring sequences in viral Ags that

regulate entry into the DRiP pathway. Results presented in the second part of this thesis

point out that an intrinsic sequence of the C-terminal p6 domain, namely the PTAP late

assembly domain, can govern protein stability, and, most importantly, the DRiP-rate of

HIV-1 Gag. Gag-DRiPs are more efficiently processed by immunoproteasomes, leading

to more efficient MHC-I presentation of Gag-derived SL (Fig. 5.15). The only marginal

difference between wt and ΔPTAP in the augmentation of SL-presentation upon IFN-γ

treatment (Fig. 5.15) indicates that the nature of Gag-DRiPs studied here might be

different to DRiPs accumulating upon IFN-γ induced oxidative stress (247).

Beyond the function in virus release, L-domains, especially the PTAP-motif of HIV-1,

have been implicated in the control of Gag ubiquitination (114, 120, 121), which could be

confirmed here using PTAP-deficient GagSL (Fig. 5.13). Nevertheless, it is not clear

Discussion

66

whether the effect of L-domains on Gag ubiquitination is related to their function to

facilitate retroviral release. Gag expressed from full-length HIV-1 seemed to be more

strongly ubiquitinated (Fig. 5.16 C). Consistently, the difference in SL-presentation

between wt Gag and the PTAP-mutant was more pronounced (Fig. 5.16 A, B). The

sensitivity of both, the increased ubiquitination (Fig. 5.13) and the enhanced SL-

presentation of the PTAP-mutant (Fig. 5.19) to overexpression of UbK48R indicates that

mutation of PTAP leads to the attachment of K48-linked poly-Ub chains to Gag. Even if

complete interference with K48-linked poly-Ub chain formation was not achieved by

overexpression of UbK48R, already a restriction in chain-length could be sufficient to

significantly reduce the efficiency of Ag processing. However, we cannot exclude that

other types of Gag ubiquitination, e.g. multiubiquitination or polyubiquitination linked

via other Lys residues of Ub, are also regulated by PTAP. The pattern of Gag

ubiquitination, also observed by others (114, 121) is consistent with multi- or

polyubiquitinated Gag species. It is tempting to speculate that monoubiquitination could

initiate further polyubiquitination of Gag. However, at least the Lys residues at position

27 and 33 of p6, which have been shown to be monoubiquitinated (111), are dispensable

for the increased Ag presentation induced by mutation of the PTAP L-domain (data not

shown). A minor fraction of GagSLΔPTAP also undergoes K63-linked polyubiquitination

(Fig. 5.13 and Fig. 5.19). As a recent publication described that K63-linked poly-Ub

chains might also represent a signal for proteasomal degradation, at least in vitro (299),

this might directly or indirectly influence the degradation of Gag by the 26S proteasome.

Interestingly, K63-linked polyubiquitination of HIV-1 Gag by HECT Ub ligases has been

recently shown to correlate with their ability to rescue virus budding (300).

The finding that mutations of PTAP result in increased Gag ubiquitination suggests that

the PTAP L-domain recruits DUBs, possibly through its interaction with the ESCRT, and

this has been proposed before (8, 114, 120, 121). The endosomal DUB AMSH interacts

with components of the ESCRT-0 and -III, deubiquitinates endosomal cargo prior to

lysosomal degradation and specifically cleaves K63-linked poly-Ub chains in vitro (301,

302). Overexpression of AMSH has been shown to block HIV-1 release, while

knockdown of AMSH had no effect on virus particle production (303, 304). However, the

L-domain dependent recruitment of AMSH to sites of viral budding and its ability to

deubiquitinate Gag in vivo have not been demonstrated.

In contrast, the presence of the PTAP motif might suppress ubiquitination by preventing

the interaction of Gag with E3 Ub ligases. It has been shown that overexpression of

Tsg101 results in enhanced ubiquitination of HIV-1 (305) and HIV-2 (306) Gag, leading

Discussion

67

to the hypothesis that Tsg101 recruits a Gag-specific E3 Ub ligase. Controversially,

disruption of the binding to Tsg101 by mutation of PTAP rather enhances the

ubiquitination of HIV-1 Gag (120, 121).

It has been proposed that the increased Gag ubiquitination observed after mutation of the

PTAP motif might not be due to sequence-specific interactions, but is rather an indirect

result of the budding defect and caused by the prolonged association with the host cell

PM (121). However, when we mutated the PTAP-motif in context of GagG2A, SL-

presentation was increased (Fig. 5.16). In addition, ubiquitinated Gag could be detected,

though the extent of Gag ubiquitination was somewhat reduced in the absence of the

myristoylation site (data not shown). These results indicate that the regulation of Gag

polyubiquitination and degradation by the proteasome is not fully dependent on its

membrane association.

The findings that interactions with the ESCRT components Tsg101 and ALIX are not

critical for directing Gag into the MHC-I pathway (Fig. 5.18), suggest that, in addition to

ESCRT interaction, the PTAP motif might regulate association of HIV-1 Gag with one or

several so far unidentified cellular factors that are connected to the cellular protein quality

system. Possible interaction partners include E3 Ub ligases, DUBs or chaperones, which

then in turn regulate the polyubiquitination and degradation of Gag during or shortly after

protein synthesis. Very recently, BAG-6, an anti-apoptotic Ub-like protein encoded

within the human MHC, has been shown to play a role in the proteasomal degradation of

DRiPs (307). It will be interesting to test the function of BAG-6 in HIV-1 infection, the

metabolism of Gag-DRiPs and MHC-I presentation of Gag-derived epitopes.

The hypothesis that PTAP acts as an autonomous stabilization signal by providing a

binding motif for cellular factors could be tested by experiments that assess of whether

regulation of MHC-I Ag presentation by PTAP works independently from its position

within Gag and is transferable to other Ags. However, the effect that PTAP exerts on

MHC-I presentation of a Gag-derived epitope might be dependent on its context within

p6. Moreover, it cannot be excluded that mutations of the PTAP motif might severely

disturb the folding and structure of Pr55, driving the protein into the DRiP-pathway.

Although the nature of DRiPs is still not fully elucidated, they are generally regarded as

erroneous, possibly misfolded byproducts of protein synthesis. Therefore, misfolded Gag-

DRiPs could interfere with virus release by disturbing the ordered assembly of Gag

molecules in a dominant-negative, prion-like manner (90).

It is tempting to speculate that this increased DRiP-rate of PTAP-mutants contributes to

the phenotype of L-domain mutants, which is characterized by a severe block in virus

Discussion

68

release and Gag processing (308, 309). Moreover, misfolding of the Gag precursor could

impair the accessibility of cleavage sites for the viral PR, contributing to the accumulation

of processing intermediates, which have recently been shown by others (310) to trans-

dominantly interfere with viral infectivity. Though it has been reported that the PTAP L-

domain is dispensable for virus release in the context of PR deficient HIV-1 (118, 309),

we observed that the PTAP motifs were necessary for efficient release of VLPs, a finding

that is in agreement with observations made by others for a comparable Gag expression

system (114).

We further wanted to evaluate the possibility that targeting HIV-1 Gag to the DRiP-

pathway by replacement of the N-terminal Met with an Arg or stable N-terminal in frame

fusion to Ub interferes with the ordered assembly, budding and release of synGag-derived

VLPs. UbRGag or UbG76VGag variants show little or no VLP-release, respectively, while

release of UbMGag-derived VLPs is only slightly attenuated (see Fig. 5.8 and Fig. 5.10).

The increased DRiP-rate of UbRGag provides a possible explanation for this effect.

However, the possible lack of myristoylation provides an alternative and more simple

explanation for the budding defect observed for Ub-Gag fusions. N-terminal Gly, a

prerequisite for myristoylation by N-myristoyltransferase (242), is exposed by processing

of the initiator Met by N-terminal Met excision (NME), catalyzed by Met

aminopeptidases (311, 312). Most likely, an N-terminal Arg in exchange for Met does not

represent a good substrate for this reaction, leading to a significant loss of Gag

myristoylation. Both, myristoylation (313) as well as cleavage of Ub (224) have been

shown to occur co-translationally. In situations where the kinetic of Ub cleavage does not

sufficiently meet that of myristoylation, this need for concerted action might explain the

slightly reduced release efficiency of the UbMGag variant.

As myristoylation is essential for targeting of Gag to the PM (6), the fraction of PM-

associated Gag was determined by membrane flotation experiments. Approximately 50 %

of wt Pr55 were found to be associated with the PM (Fig. 5.11), which is in agreement

with comparable studies performed in HeLa cells (314-316). However, the membrane-

bound Gag fraction can differ considerably, depending on the cell type (317). In contrast

to UbMGag, whose membrane association was found to be only slightly reduced, both

UbRGag and UbG76VGag failed to associate with the PM as shown by comparison to the

myristoylation-deficient GagG2A mutant.

Though certain differences between Gag variants with respect to subcellular distribution

could be observed by immunofluorescence analysis using a CA-specific Ab (Fig. 5.11 A-

D), the overall staining pattern was quite diffuse, similar to Gag-expressing EL4 cell lines

Discussion

69

(data not shown; see (136)). Such a pattern has been found in some studies for

unprocessed Gag (318, 319), but is in contrast to most reports (7, 320-322), which

described a more punctate pattern or increased fluorescence intensity at the PM. The

discrepancies concerning Gag distribution might be explained by differences in

expression levels, as noticed by others (318), or staining procedures. In addition, it has

been suggested previously that the route of mRNA export from the nucleus might

contribute to Gag localization (323, 324). However, to which extent this applies to codon-

optimized, Rev-independent Gag expression systems is not clear.

Despite the insensitivity of HIV-1 Gag to the N-end rule in terms of overall protein half-

life (210), it is demonstrated in this work that targeting of HIV-1 Gag into the DRiP-

pathway, either by an N-terminal Arg, stable N-terminal fusion of Ub or by mutation of

the PTAP-motif, correlates with an increase in MHC-I Ag presentation.

The MHC-I pathway is considered as a complex and multi-step process. We analyzed the

initial step, the synthesis and degradation of an Ag, and the final step, the amount of

pMHC-I complexes on the cell surface. Since the amount of total H2-Kb-molecules and

the rate of reappearance after the acid wash procedure were similar in both EL4 cell lines,

we assume that the H2-Kb expression levels as well as the intermediate steps in the MHC-

I pathway do not markedly differ between the two EL4 cell lines. The differences in the

proportion of H2-Kb-molecules loaded with SL epitope, however, should be consequent

to disparities in the DRiP-rate. Similar to previous studies (133, 211, 228, 232-236), the

generation of the SL epitope was dependent on proteasome activity and ongoing protein

biosynthesis, further supporting the notion that this phenomenon studied in our system is

related to the DRiP pathway.

The increased SL-presentation observed for the UbRGagSL/EL4 cell line could be

reproduced in transiently transfected human HeLa-Kb cells. Interestingly, targeting Gag

for degradation by the UFD pathway resulted in an even more dramatic increase in SL-

presentation, again without significantly affecting the overall metabolic half-life of Gag.

It may be interesting to test the immunogenicity of this UbG76VGag variant in further in

vivo studies.

The budding defect caused by mutation of the PTAP-motif might lead to the intracellular

accumulation of Gag, thus increasing the availability of Ag for processing by the 26S

proteasome. This would provide an obvious explanation for the better entry of PTAP-

deficient Gag into the MHC-I pathway. However, we could exclude that there is a

correlation between budding capacity and MHC-I Ag presentation. Impairment of VLP-

release by mutation of the myristoylation site (G2A; Fig. 5.17), knockdown of Tsg101 or

Discussion

70

overexpression of ALIX (Fig. 5.18), all three known to induce a budding defect (4, 11,

26), did not lead to an increase in SL-presentation. Consistently, rescue of the budding

defect of GagΔPTAP by ALIX overexpression did not reduce SL-presentation (Fig. 5.18).

Data presented in this thesis strongly suggests that targeting of a given Ag into the DRiP-

pathway represents a suitable strategy to augment the number of pMHC complexes at the

cell surface. Most importantly, the increase in MHC-I Ag presentation by N-end rule

targeting of Gag was accompanied by enhanced T-cell activation, both, in vitro and in

vivo (Fig 5.6 and Fig. 5.7). A correlation between the amount of TCR-ligands at the

surface of professional Ag presenting cells (pAPCs) and the induction of a CTL response

has been shown in other models (246, 325-327). However, in some studies, memory

CTLs of lower avidity were generated (325) or progressively declined in number (246).

Nevertheless, the specificity of CTLs seems to be more crucial for their ability to kill

HIV-1-infected target cells compared to their functional avidity (328). Therefore, it will

be important in the future to analyze not only the artificially introduced SL epitope, but

also the MHC-I presentation of genuinely Gag-derived epitopes, for example the HLA-

A2 restricted MA-derived SLYNTVATL.

Targeting of Ags for proteasomal degradation by the N-end rule or UFD pathway has

been employed in several DNA immunization studies. N-end ruled variants of β-Gal

(288) or HIV-1 Nef (206) induced more frequent CTL responses when delivered into

mice using plasmids or rVV vectors. The N-end rule targeting of HIV-1 Pol increased

MHC-I presentation by human DCs in vitro and CTL responses in humanized mice (329).

Enhanced and protective CTL responses have been successfully induced by UbG76A

fusions to the E6 protein of cottontail rabbit papillomavirus (284), to the lymphocytic

choriomeningitis virus (LCMV) NP (287), to HIV-1 subgenomic sequences (330), to

EBV latent membrane protein 11 (286) and Trypanosoma cruzi amastigote surface

protein-2 (331).

Wong et al. reported that rVV-mediated expression of sub-genomic, altered and highly

turned over fragments of Gag resulted in increased numbers of SL-H2-Kb complexes

presented at the cell surface (210). However, when CTL responses were compared in

DNA vaccination studies in mice, T-cell responses in vivo did not correlate with the Ag

processing rates of those Gag variants. Variations between full length wt Gag studied here

and fragments of Gag studied by others (210) might contribute to those differences.

Consistently, in these reports, highly efficient induction of a protective CTL response was

observed in the virtual absence of a humoral immune response. For example, N-end rule

or UFD targeting of IAV NP induced lower Ab responses while CTL responses directed

Discussion

71

against a specific epitope were comparable to wt NP (293). The reduced humoral immune

response is thought to be a consequence of reduced availability of the full-length Ag,

presumably due to increased continuous turnover and reduced steady-state levels. Rapidly

degraded forms of Ag may not only fail to elicit Ab responses, but some studies find a

clear correlation between metabolic stability of the vaccine Ag and induction of a CTL

response (332-334). This phenomenon can be explained by the assumption that rapidly

degraded forms of Ag represent poor substrates for cross-presentation, which is thought

to be an important mechanism for activation of naïve CD8+ T cells in vivo (140, 335-338).

This process, called cross-priming, takes place when pAPCs take up exogenous Ag

transferred, for example, from virus-infected or tumor cells. Cross-priming might be

especially, but not exclusively, important for induction of CTL responses against viruses

that do not preferentially infect pAPCs.

Particulate Ags are introduced into pAPCs by phagocytosis, whereas soluble Ags can be

taken up via receptor-mediated endocytosis or pinocytosis. Interestingly, transfer of

peptides through gap junctions has also been described (339). DCs can also acquire

preformed pMHC complexes by trogocytosis (340, 341). This term describes the

exchange of PM patches between cells, enabling the intercellular transfer of membrane

proteins (342). As ubiquitinated Gag associates with membranes (121) and proteasome

inhibitors cause the accumulation of Gag-DRiPs in insoluble protein aggregates

(unpublished observation), driving Gag into the DRiP-pathway might also enhance its

attraction for cross-presentation.

Although CD8+ T cells generally exhibit an exceptional high sensitivity for specific

pMHC-I complexes (343), enhancing Ag processing can be advantageous in certain

situations. First, enhancing the MHC-I processing of viral Ags might be important for the

recognition of virus-infected cells during the onset of synthesis of viral proteins,

especially of stable, structural proteins like the retroviral Gag polyprotein (129, 344).

During peptide loading of MHC-I molecules in the ER, a great number of peptides,

derived from turnover of an estimated number of 2-3x109 cellular proteins compete for

the available number of binding grooves (129). Thus, an enhanced DRiP-rate should

result in an increased generation and steady-state level of antigenic peptides. However, an

emerging concept of compartmentalization of protein synthesis and Ag processing

suggests that the law of mass action could be circumvented during peptide generation and

loading (reviewed in (345)). In addition, at early stages of virus infection, only few target

cells may be available for recognition by CTLs. In the in vivo model of adoptive transfer,

2x106 of both target cell lines, UbRGagSL/EL4 and UbMGagSL/EL4, were sufficient to

Discussion

72

induce maximal stimulation of 1x107 co-transferred SL-specific OT-1 T cells. However,

when the number of target cells was limited to 2x105, a clear difference in OT-1 T-cell

proliferation was observed. This indicates that for efficient stimulation of CD8+ T cells in

vivo, limited availability of target cells can be compensated by a higher presentation rate

of specific pMHC-I complexes per target cell.

Second, CD8+ T cells compete with each other for pMHC-I complexes at the surface of

target cells in a process called cross-competition, which is especially important to shape

the hierarchy of T-cell responses (346). Increased numbers of specific pMHC-I

complexes might therefore help to induce CTLs of the desired specificity.

Third, enhanced MHC-I presentation of epitopes derived from tumor Ags seems

favorable to break tolerance and can initiate an anti-tumor CTL response (347-349).

An elegant study conducted by the group of Nilabh Shastri recently characterized DRiPs

derived from EBV EBNA-1 as truncated polypeptides, whose synthesis is governed by

the GA-stretch of the protein (298). The authors speculated that herpesviral proteins

involved in episome maintenance might have evolved to interfere with the formation of

DRiPs and, thus, with MHC-I Ag presentation. The novel function of PTAP in regulation

of DRiP-formation might also decrease MHC-I presentation of Gag-derived epitopes in

vivo and, thus, contribute to immune escape during HIV-1 infection. Cryptic epitopes

derived from HIV frameshift sequences are presented to CTLs in infected individuals

(350). Those polypeptides might be regarded as nonsense products of incorrect reading

frame selection by the ribosome and could therefore be considered as DRiPs. In addition,

APOBEC3 editing of HIV, which is known to produce truncated and misfolded viral

proteins, enhances CTL recognition of infected cells (351). Further research is needed to

elucidate the nature of DRiPs derived from HIV-1 proteins, their contribution to the pool

of MHC-I-presented epitopes and countermeasures that HIV-1 might have evolved.

Taken together, data presented in this thesis support the hypothesis that DRiPs constitute

the main source for endogenous peptides presented by the MHC-I pathway. Moreover, it

was shown that HIV-1 Gag can be targeted into the DRiP-pathway either by artificial

introduction of degradation signals or by mutation of the PTAP L-domain motif within

the p6 domain. These findings could be interesting with respect to new ideas for the

design of optimized vaccine Ags.

Material and methods

73

7 Material and methods

Expression plasmids, siRNA and molecular cloning

The expression plasmid psyngag encoding the entire Pr55 Gag polyprotein originating

from the isolate HIV-1HX10 as well as the construction of the plasmids pUbMsyngag and

pUbRsyngag have been described elsewhere (136, 218). For pUbG76Vsyngag, the C-

terminal Gly residue of the Ub-fusion part was mutated to Val using the QuikChange site

directed mutagenesis kit (Stratagene) and oligonucleotides (Biomers) G76V-fw (5´-TCC

TGC GCT TGA GGG GGG TGA TGG GCG C-3´) and G76V-rc (5´-GCG CCC ATC

ACC CCC CTC AAG CGC AGG A-3´).

The introduction of the SL-coding sequence into psyngag as well as generation of a

minigene expression construct encoding MSIINFEKL have been described before (136).

Both copies of the PTAP motif have been exchanged for the sequence AIVA by site-

directed mutagenesis using the QuikChange kit (Stratagene) and oligonucleotides

PTAP1-fw (5´-GCA GGC CCG AGG CCA TCG TCG CCC CCT TCC TGC-3´) and

PTAP1-rc (5´-GCA GGA AGG GGG CGA CGA TGG CCT CGG GCC TGC-3´) as well

as PTAP2-fw (5´-GGC CCG AGG CCA TCG TCG CCC CCG AGG AGA-3´) and

PTAP2-rc (5´-TCT CCT CGG GGG CGA CGA TGG CCT CGG GCC-3´). To introduce

the SL-coding sequence into the proviral constructs pNL4-3 (248), pBRNL4-3nef-IRES-

GFP (249) and the HIV-1 NL4-3 subgenomic expression vector pNLenv1, in which the

env gene is deleted (250, 352), as well as mutants thereof (ΔPTAP) in which the PTAP-

motif has been exchanged for LIRL without affecting the overlapping pol reading frame

(309), the gag gene was subcloned into the pGEM-T vector (Promega) via Sph I and Sbf

I. A BstE II site was introduced by site-directed mutagenesis using oligonucleotides 5´-

GAA GCA ATG AGC CAG GTG ACC AAT CCA GCT ACC-3´ and 5´-GGT AGC

TGG ATT GGT CAC CTG GCT CAT TGC TTC-3´. Oligonucleotides BstEII-SL-fw (5´-

GTG ACC TCG ATC ATC AAC TTC GAA AAG CTA-3´) and BstEII-SL-rv (5´-GTC

ACT AGC TTT TCG AAG TTG ATG ATC GAG-3´) were used to introduce the SL

epitope and gag was cloned back using Sph I and Sbf I. All sequences were confirmed by

DNA sequencing using the Big Dye v3.1 sequencing kit (Applied Biosystems) on an ABI

PRISM 3100 sequencing instrument (Applied Biosystems).

The expression vector pHA-Ub has been obtained from H.-G. Kräusslich and is described

elsewhere (114). Construction of pCMV-FLAG-ALIX is described elsewhere (353).

Material and methods

74

For knockdown of Tsg101, synthetic siRNA (Integrated DNA technologies) was used as

described (11). The sequence was sense 5´-CCU CCA GUC UUC UCU CGU CTT-3´and

antisense 5´-GAC GAG AGA AGA CUG GAG GTT-3´.

Cell culture, transfection procedure and generation of stable cell lines

EL4, B3Z and Jurkat T cells, all growing in suspension were maintained in RPMI 1640

medium supplemented with 10 % (v/v) heat-inactivated FCS, 2 mM L-glutamine,

100 U/ml penicillin, 100 µg/ml streptomycin, 0.01 % sodium pyruvate and 0.1 %

nonessential amino acids. EL4 is a thymoma cell line derived from the C57BL/6 mouse

(H2-Kb). The EL4-derived cell line E.G7 synthesizes and secretes OVA (239). The SL-

H2-Kb-specific, murine CD8+ hybridoma T cells B3Z express the lacZ reporter gene

under the control of the NFAT enhancer (354). The Jurkat T cell leukemia line was

isolated from blood and was initially called JM. (355). The generation of stable gag-

expressing EL4 cell lines is described elsewhere (136).

HeLaSS6, HeLa-TZM-bl (356) and 293T cells were cultured in Dulbecco´s modified

Eagle medium (DMEM) supplemented with 10 % (v/v) inactivated fetal calf serum

(FCS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. For HeLa-

Kb cells (241), which express high levels of H2-Kb and were obtained with permission of

Ian York, Michigan State University, 1 mg/ml of G418 were added. All media and

compounds were purchased from Gibco. Transfections were performed using

Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

p24 antigen ELISA

Quantification of p24 Ag was performed by enzyme-linked immunosorbent assay

(ELISA; Aalto) according to the manufacturer’s instructions. Deviations from the

standard protocol for determination of Gag expression in EL4 cells are described

elsewhere (136).

Single round infection assay

In 96 well plates, HeLa TZM-bl indicator cells were infected in triplicates

(4000 cells/well) with 2 ng of p24 in a volume of 100 µl medium containing 10 μg/ml

polybrene. To prevent further spread of infection, fresh medium containing 100 µg/ml

dextran sulphate was added after overnight incubation. 48 h postinfection, cells were

washed with PBS and lysed by addition of Tropix® Gal-Screen® substrate (Applied

Biosystems, buffer B). Lysates were transferred into opaque 96 well plates (Corning

Material and methods

75

Costar) and incubated at 28 °C for 1 h. Luminescence was measured in an ELISA reader

(Bio-Tek).

Viruses

Cell culture supernatant was harvested 48 h after transfection of 293T cells with proviral

constructs and passed through a 0.45 µm pore-size filter. Virus was pelleted through 20 %

(w/v) sucrose (16000 x g, 4 °C, 90 min) and stocks were normalized for p24 as quantified

by p24 ELISA. Aliquots were stored at -80°C.

Infection of T cell lines

For infection of T cell cultures, 1×105 Jurkat T cells were incubated overnight with 20 ng

of p24, and cell culture supernatant was collected every third day postinfection (dpi).

Virus replication was assessed by quantification of the virus-associated RT activity by

[32P]-TTP incorporation using an oligo(dT)-poly(A) template as described elsewhere

(357).

Flow cytometry

For detection of H2-Kb-bound SL-epitope or H2-Kb molecules, cells were incubated with

hybridoma cell culture supernatant containing the monoclonal antibodies (mAbs)

25D1.16 (227) or B8-24-3 (358), respectively, followed by staining with secondary

chicken anti-mouse-AlexaFluor647 Ab (Invitrogen). Alternatively, 25D1.16 mAb was

purified from hybridoma cell culture supernatant and labeled with an AlexaFluor647

labeling kit as described in (136), or obtained as an allophycocyanin (APC)-conjugate

from a commercial source (25D1.16-APC, eBioscience). H2-Kb-SL complexes derived

from NL4-3Δenv were detected using Phycoerythrin-conjugated 25D1.16 (25D1.16-PE;

eBioscience) diluted 1:100 in FACS buffer (5 % (v/v) FCS, 0.02 % (v/v) NaN3 in PBS).

Intracellular Gag was detected by staining with a FITC-conjugated anti-p24 Ab (KC57-

FITC; Beckman Coulter) diluted 1:100 in Perm/Wash™ buffer (BD Biosciences) after

permeabilization of cells using Cytofix/Cytoperm™ (BD Biosciences). Flow cytometry

was performed on a FACSCalibur using CellQuest software (BD Biosciences). Data

analysis was performed using the FCS Express V3 software (De Novo).

Acid wash

SL-H2-Kbcomplex formation at the cell surface was followed by flow cytometry after an

acid wash procedure. Cells were incubated for 2 min at pH 3 and 4 °C in buffer

Material and methods

76

containing 131 mM sodium citrate and 66 mM NaH2PO4. After neutralization in PBS,

cells were incubated in complete RPMI medium in the presence or absence of 20 µM

carbobenzoxyl-leucine-leucine-leucinal (zLLL; Sigma) or protein synthesis inhibitors

(PSIs; 6.3 µM emetine, 8.3 µM cycloheximide and 1.7 µM puromycin; all purchased

from Sigma) for desired periods at 37 °C. At least 500,000 cells were incubated with

supernatants of hybridomas 25D1.16, B8-24-3 or medium alone as a control for 1 h on

ice, followed by two washing steps in PBS and staining with Cy2-conjugated anti-mouse

IgG (Rockland) diluted 1:200 in FACS buffer for 30 min on ice. Cells were fixed and

analysed by flow cytometry as described above.

Immunocytochemistry

For immunofluorescence analysis, cells were seeded onto coverslips (Superfrost, Roth),

transiently transfected and fixed the next day for 30 min at room temperature (RT) in 3 %

paraformaldehyde (PFA). Following permeabilization in 0.1 % (v/v) Triton X-100 in

PBS, cells were washed twice and blocked for 30 min in 1 % (w/v) BSA (bovine serum

albumin) in PBS. Staining was performed for 20 min at RT using FITC-conjugated anti-

p24 Ab (KC57; Beckman-Coulter) diluted 1:200 in 1 % BSA in PBS and 1 µg/ml of

DAPI (4’,6-Diamidin-2’-phenylindoldihydrochlorid, Pierce) for 10 min to counterstain

nuclei, followed by three washing steps in PBS. Immunofluorescence was visualized

using a confocal Leica TCSSP5 microscope DMI 6000.

Time course analysis of Gag synthesis and budding

For analysis of Gag synthesis, 0, 4, 6, 8 and 12 h after transfection of HeLa cells with

various psyngag expression plasmids, cells were harvested and lysed in CHAPS-

deoxycholate lysis buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5 % (w/v) CHAPS

(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate), 0.3 % (w/v) sodium

deoxycholate) or RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 % (v/v)

Nonidet P-40, 0.5 % (w/v) sodium deoxycholate, 0.1 % (w/v) SDS, 5 mM EDTA)

supplemented with 1 mM phenylmethylsulphonylfluoride (PMSF), 5 mM N-

ethylmaleimide (NEM), 20 μM zLLL and complete protease inhibitor cocktail

(Boehringer, Mannheim). Equal amounts of soluble protein as quantified by bicinchoninic

acid (BCA) assay (Pierce) were subjected to SDS-PAGE and Western blotting.

For analysis of VLP release, cells were aliquoted 24 h after transfection and, after 0, 0.5,

1, 2 and 4 h, cells and supernatants were collected by centrifugation. VLPs released into

the cell culture supernatant were pelleted through 20 % (w/v) sucrose in PBS by

Material and methods

77

centrifugation and directly lysed in 2x SDS sample buffer (125 mM Tris-HCl, pH 6.8,

4 % (w/v) SDS, 20 % (v/v) glycerol, 10 % (v/v) ß-mercaptoethanol or 100 mM

dithiothreitol (DTT), 0.02 % (w/v) bromphenol blue).

Western blotting and antibodies

Proteins separated by SDS-PAGE were blotted onto polyvinylidenefluoride (PVDF)

membranes (HybondTM, GE healthcare) and incubated with a specific primary Ab

followed by incubation with horseradish peroxidase (HRP)-conjugated secondary Abs

(Dianova) if required and standard enhanced chemiluminescence (ECL) procedure.

The following antibodies or antisera were used at the indicated dilutions in low fat milk

solution or 1 % BSA in PBS/0.1 % Tween: rabbit anti-p6 antiserum (1:10,000; (359),

rabbit anti-p24 antiserum (1:10,000; Seramun), monoclonal anti-β-actin Ab (1:10,000;

Sigma), monoclonal anti-Transferrin receptor Ab (TfR; 1:1000, Zymed), pooled serum of

20 HIV-1 positive patients (PKT, 1:5,000, NIH AIDS Research Reference Reagent

Program, USA, Cat.Nr. 3975 or pooled sera obtained from the Nationales

Referenzzentrum für Retroviren, Institute for Clinical and Molecular Virology,

Universitätsklinikum Erlangen, Germany), monoclonal rat anti-Hemagglutinin Ab (HA;

clone 3F10, HRP-conjugated, 1:10,000; Roche), ribosomal P antigen (RP0)-specific

antiserum (1:5,000; Immunovision Inc.), monoclonal anti-β5i (LMP7) Ab (1:1,000; Enzo

Life Sciences), polyclonal rabbit anti-β5 (1:1,000; Enzo Life Sciences).

Pulse-chase analysis

Pulse-chase experiments were basically performed as described elsewhere (133, 136). For

detection of Gag-DRiPs, short-term pulse-chase experiments were performed, whereas

long-term pulse-chase experiments were used to analyze protein stability. Briefly, for

short-term pulse-chase experiments, transgenic EL4 cells ore HeLa cells were washed in

PBS and treated with 20 µM of each zLLL and LC or DMSO as solvent control during

the last 10 min of a 30 min starvation period in Met-free, serum-free RPMI (Invitrogen).

Cells were radiolabelled for 15 min (EL4 cells) or 3 min (HeLa cells) with 3 mCi/ml

[35S]Met (Amersham Life Sciences or Hartmann Analytic) and chased for up to 120 min

while shaking at 37 °C in DMEM supplemented with 10 % fetal calf serum and 10 mM

Met in the presence or absence of PIs. Cells were harvested and lysed in 200 µl CHAPS-

Doc or RIPA buffer supplemented with 1 mM PMSF, 5 mM NEM and complete protease

inhibitor cocktail for 5 min on ice and separated from the insoluble fraction by

centrifugation at 20,000x g for 10 min. Gag was recovered by immunoprecipitation (IP)

Material and methods

78

using a mixture of polyclonal rabbit anti-p6 and anti-p24 antibodies or human PKT

prebound to Protein G-Sepharose (GE Healthcare). Samples were separated by SDS-

PAGE on a 10 % (w/v) ProSieve gel (Cambrex Bioscience) backed with Gel Bond film

(FMC Bioproducts). Following fixation for 1 h in 40 % methanol, 10 % acetic acid, gels

were rinsed with water, soaked in 1 M sodium salicylic acid solution for 5 h, and dried.

Radioactivity in gels was analyzed using phosphorimaging instrumentation (Fujifilm

BAS-2000, Fujifilm) or fluorography using BioMax MR films (Kodak) and quantified by

AIDA imaging software (Raytest).

For long-term pulse-chase experiments, after a 30 min starvation period, cells were

radiolabelled with 3 mCi/ml [35S]Met for 30 min (EL4 cells) or 10 min (HeLa cells),

plated in DMEM containing an excess of unlabeled Met and chased for up 48 h. Analyses

of radioactivity in cell lysates were performed as described above.

Adoptive transfer of OT-1 T cells and INF-γ ELISPOT

Adoptive transfer of OT-1 T cells and INF-γ ELISPOT after immunization of naïve

C57BL/6 mice with GagSL expressing were performed by Dr. A. Goldwich as described

in (136). Briefly, spleen and LN cells from OT-1 mice (360), were labeled with CFSE

(Carboxyfluorescein succinimidyl ester, Vybrant CFDA SE Kit; Molecular Probes) and

transferred i.v. into the tail vein of naïve C57BL/6 recipient mice (10 x 106 cells/mouse,

Charles River Laboratories). Parental or transgenic EL4 cells (0.2 x 106 cells/mouse) were

transferred 5 min later into the contralateral tail vein. Animals were sacrificed two or

three days after adoptive transfer, spleen cells were harvested and analysed by flow

cytometry. In order to analyze the OT-1 cell proliferation, OT-1 cells were identified

within the live lymphocyte (FSC/SSC) gate by staining for the transgenic T cell receptor

(TCR) α-chain Vα2, and CFSE levels were quantified.

Naïve C57BL/6 mice were injected into the tail vein with parental EL4 or UbGagSL/EL4

cells. As a positive control, 50 µg of synthetic SL-peptide dissolved in 50 µl incomplete

Freunds adjuvant were subcutaneously injected near the tail root. After nine days

splenocytes were collected and an ELISPOT assay was performed. Briefly, in 96-well

ELISPOT plates precoated with rat anti-mouse IFN-γ antibody (MabTech). Following

washing, splenocytes (10x106 cells/ml) were added in triplicates and cells were pulsed

with SL peptide (100 ng/ml) or phytohemagglutinin (PHA) and staphylococcal

enterotoxin A (SEA; 5 μg/ml each) in 100 µl RPMI-1640 medium supplemented with

10 % (v/v) FCS. To control for unspecific reaction, cells were treated in parallel without

peptide. After incubation at 37 °C in a 5 % CO2 atmosphere for 20 h, cytokine production

Material and methods

79

was detected by using biotinylated Ab against IFN-γ (5 μg/ml, MabTech, Hamburg) and

alkaline phosphatase-streptavidin (0.2 U/ml). The IFN-γ spots were developed by

addition of 100 μl of BCIP/NBT solution. Spots in dried plates were counted using

computer-assisted image analysis with a Carl Zeiss Axioplan 2 and VisionKS ELISPOT

version 4.9.15. For analysis, the number of spots without peptide (unspecific reaction)

were subtracted from the number of spots with peptide (specific reaction). To control for

variations in the frequencies of CD8+ cells in the spleens of individual mice, FACS

analyses using anti-CD8a-FITC (Beckmann Coulter) were performed.

Transmission electron microscopy (TEM)

For TEM, HeLa cells were transfected with individual psyngag expression plasmids and,

24 h posttransfection, transferred into cellulose capillary tubes (361) and grown for

another 24 h. Following fixation in 3 % paraformaldehyde for 1 h at 37 °C, capillaries

were stored until further preparation at 4°C.

Tubes were collected by centrifugation and sealed by immersion in low-melting-point

agarose. The samples were post fixed with OsO4 (1% in distilled water, 1h), tannic acid

(0.1 % in Hepes 0.05 M, 30 min) and uranyl acetate (1 % in distilled water, 2h) followed

by stepwise dehydration in graded ethanol and embedding in epon resin, which was

subsequently polymerized. Thin sections were prepared with an ultramicrotome (Ultracut

S; Leica,) and counterstained with uranyl acetate and lead citrate. The sections were

examined using a TEM 902 (Carl Zeiss SMT AG) at 80 kV, and the images were

digitized using a slow-scan charge-coupled-device camera (Pro Scan).

Membrane flotation by Optiprep density gradient centrifugation

HeLa cells were transiently transfected with psyngag expression plasmids, washed twice

in PBS and subsequently detached in ice-cold PBS containing 10 mM EDTA. Following

one more washing step in PBS, cells were washed in homogenization buffer (0.25 M

sucrose, 1 mM EDTA, 2 mM MgCl2, 20 mM Hepes-NaOh, pH 7.4), centrifuged and

resupended in 500 µl of homogenization buffer. Cells were disrupted by sonfication in ice

water and the homogenate was centrifuged at 2000 x g for 12 min to remove unbroken

cells and nuclei. The supernatant was adjusted with Optiprep (Progen) to final

concentration of 30 % iodixanol.

In SW41 centrifuge tubes, a discontinuous Optiprep gradient, containing, from top to

bottom, 2.5 %, 10 %, 17.5 % and 25 % of iodixanol diluted in 0.25 M sucrose, 6 mM

Material and methods

80

EDTA, 12 mM MgCl2, 120 mM Hepes-NaOh, pH 7.4 was prepared and finally

underlayered with 600 µl of the sample.

Following centrifugation for 5 h at 151,000 x gav in a SW41 rotor (Beckman Coulter),

material above the visible membrane-containing fraction was displaced, fractions à 0.5 ml

were collected from top of the gradient, boiled in 2x SDS sample buffer and subjected to

SDS-PAGE and Western blotting using Abs specific for p6, TfR and RP0. Localization of

Gag in membrane fractions was calculated after densitometric quantification as the

amount of Gag present in membrane fractions (containing TfR, but not RP0) divided by

the total amount of Gag.

Statistical analysis

Mean fluorescence intensities (MFIs; geometric mean) obtained in flow cytometry

experiments were compared using the Mann-Whitney U test (two-tailed, P ≤ 0.05). Data

obtained in individual pulse-chase experiments were subjected to regression analysis,

from which the mean metabolic half-lives of GagSL variants were calculated and

compared by a two-tailed t-test.

References 81

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Acknowledgements

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9 Acknowledgements

I want to thank for their help and support during the preparation of this thesis:

• Prof. Dr. Ulrich Schubert for his guidance, critical advice, and discussions. I thank him for his constant support, and for patiently teaching me scientific writing. I especially appreciate that he provided the freedom to work independently and to enjoy the creativity of scientific work.

• Prof. Dr. Bernhard Fleckenstein for the possibility to prepare this thesis at the Institute of Clinical and Molecular Virology, which provides not only a vivid scientific environment, but also a wonderful location.

• All colleagues that are currently or formerly working on the Gag-DRiP project:

o Andreas Goldwich, from whom I have learned much about (techniques in) protein biochemistry and immunology, for initiating fruitful cooperations and performing laborious in vivo experiments.

o Sandra Schreiber for the wonderful teamwork, especially during pulse-chase and extended time-course experiments.

o Stefanie Meier for technical assistance.

o Christian Setz, who is a very skilled experimentator and a reliable person, for his constant help and the considerably contribution to this project.

o Julia Wild and all other students who have worked on this project.

• All other current and former members of the lab of Ulrich Schubert, especially Jörg Votteler, Friedrich Hahn, André Eißmann and Stefan Sörgel, for help, sharing of reagents and knowledge, for support, critical reading, discussions and a lot of fun.

• All members of the Institute of Clinical and Molecular Virology for their support.

• Prof. Dr. Ralf Wagner and his group for kindly providing the syngag constructs.

• Our cooperation partners from the department of Dermatology: Prof. Dr. Manfred Lutz, Prof. Dr. Eckhardt Kämpgen, Dr. Jan Dörrie, Dr. Niels Schaft and Christian Hofmann.

• Dr. Peter Henklein and René Röder for peptide synthesis.

• Dr. Norbert Bannert for electron microscopy.

• Dr. Victor Wray and Helga Litschl for critical reading.

• All other people sharing reagents or good ideas.

• Prof. Dr. Robert Slany for reviewing this thesis.

• My friends, especially Stefanie Buerbank, for always lifting my spirits.

• Dieter Norkauer for being by my side for the last 8 years. I thank him for offering a shoulder to lean on, for his continued support, sympathy and encouragement.

• And, most importantly, my parents, who decidedly played a role in where I am today.

C U R R I C U L U M V I TA E SABINE HAHN

AUSBILDUNG Studium: 10/2000 – 08/2005 Diplomstudiengang Molekulare Medizin

Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg

Hauptfach: Immunologie

Nebenfächer: • Entwicklungsbiologie und Embryologie • Pharmakologie und Toxikologie • Neurowissenschaften

Abschluss: Diplom (Note: 1,1)

01/2005 – 08/2005 Diplomarbeit am Institut für Klinische und Molekulare Virologie

der FAU Erlangen-Nürnberg

Thema: Untersuchungen zur Korrelation von Proteinstabilität und MHC-I Antigenpräsentation am Beispiel des HIV-1 Strukturproteins Gag.

Betreuer: Prof. Dr. Ulrich Schubert Schulbildung: 09/1991 – 06/2000 Werner-von-Siemens-Gymnasium in Regensburg

Abschluss: Allgemeine Hochschulreife (Note: 1,0) STIPENDIEN 09/2005 – 01/2006 Doktorandenstipendium des Graduiertenkollegs 1071 „Viren des

Immunsystems“ 10/2000 – 03/2005 Stipendium für besonders Begabte nach dem Bayerischen

Begabtenförderungsgesetz PRAKTISCHE ERFAHRUNGEN 09/2005 – 07/2011 Promotionsarbeit am Institut für Klinische und Molekulare

Virologie der FAU Erlangen-Nürnberg im internationalen Doktorandenkolleg „Leitstrukturen der Zellfunktion“ des Elitenetzwerks Bayern

Thema: Die Bedeutung fehlerhafter ribosomaler Produkte für die MHC Klasse I Antigenpräsentation des humanen Immundefizienzvirus-1 Strukturproteins Gag.

Betreuer: Prof. Dr. Ulrich Schubert

07/2004 – 09/2004 Laborpraktikum im Bereich Neuro- und Zellbiologie an der Ecole Normale Supérieure in Paris

Institut national de la santé et de la recherche médicale unité 497 „Biologie de la synapse et régulation de la survie neuronale”

Betreuer: Dr. Christian Vannier 04/2004 – 05/2004 Laborpraktikum im Bereich Immunologie und Zellbiologie am

Institut für Molekulare Immunologie der FAU Erlangen

Betreuer: Dr. Reinhard Voll/Prof. Dr. Hans-Martin Jäck 06/2003 – 12/2003 und Studentische Hilfskraft am Institut für Medizininformatik, 06/2002 – 09/2002 Biometrie und Epidemiologie der FAU Erlangen 07/2001 – 10/2001 Studentische Hilfskraft am Institut für Biochemie der FAU

Erlangen 03/2001 – 04/2001 Praktikum am nationalen Referenzzentrum für humane

spongiforme Enzephalopathien, Universitätsklinikum Göttingen

Betreuer: Dr. Inga Zerr PUBLIKATIONEN • Hahn S., Setz C., Wild J., Schubert U. The PTAP sequence within the p6 domain of

human immunodeficiency virus type 1 Gag regulates its ubiquitination and MHC class I antigen presentation. Journal of Immunology 86(10):5706-18.

• Votteler J., Neumann L., Hahn S., Hahn F., Rauch P., Schmidt K., Studtrucker N., Solbak S.M., Fossen T., Henklein P., Ott D.E., Holland G., Bannert N., Schubert U. (2011) Highly conserved Serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly. Retrovirology 8 (11).

• Goldwich A., Hahn S., Schreiber S., Meier S., Kämpgen E., Wagner R., Lutz M.B., Schubert U. (2008) Targeting HIV-1 Gag into the defective ribosomal product pathway enhances MHC class I antigen presentation and CD8+ T cell activation. Journal of Immunology 180(1):372-82.

• Meister S., Schubert U., Neubert K., Herrmann K., Burger R., Gramatzki M., Hahn S., Schreiber S., Wilhelm S., Herrmann M., Jack H.M., Voll R.E. (2007) Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition. Cancer Research 67(4):1783-92.