Cytomegalovirus/entry/–lessons/learned/ from/the/behavior ...
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Cytomegalovirus entry – lessons learned from the behavior of gH/gL envelope glycoprotein complex Marija Backovic Félix Rey Unité de Virologie Structurale InsGtut Pasteur, Paris David Veesler, University of Washington, SeaLle, USA
Transcript of Cytomegalovirus/entry/–lessons/learned/ from/the/behavior ...
Cytomegalovirus entry – lessons learned from the behavior of gH/gL envelope
glycoprotein complex
Marija Backovic
Félix Rey Unité de Virologie Structurale
InsGtut Pasteur, Paris
David Veesler, University of Washington, SeaLle, USA
Herpesviruses – enveloped, DNA viruses
Family Herpesviridae:
• more than 200 members
• infect vertebrates and invertebrates
• 8 infect humans
Genome:
• linear, double-‐stranded DNA
• 120 to 230 kb (70 to 200 genes )
Lipid membrane
DNA genome (capsid)
100nm
Herpes Simplex Virus 1 (HSV-‐1)
100nm
Human herpesviruses / life-‐long infecGons
Sub family"
Name (abbrevia;on)"
α"
Herpes Simplex 1 (HSV-‐1)
Herpes Simplex 2 (HSV-‐2)
Varicella-‐Zoster (VZV)"
β"
Cytomegalovirus (CMV)"
Herpes lymphotropic virus (HHV-‐6)"
Human herpes virus-‐7 (HHV-‐7) "
γ"
Epstein-‐Barr Virus (EBV) "
Kaposi's sarcoma-‐associated herpes virus (KSHV)
1° InfecGon
LyGc replicaGon Latency
Transmission
CMV • Leading cause of congenital birth
defects • ProblemaGc for paGents with
suppressed immune system
Herpesviruses – structural organizaGon of the virion
Proteome analysis: 35-‐45 major species
Envelope: 4-‐19 glycoproteins involved in entry
Tegument: 9-‐17 accessory proteins
Capsid: DNA genome + 4-‐7 capsid proteins
From Grunewald K. et al, Science 2003
100nm
Merger of viral and cell membranes
Entry of herpesviruses is mediated by a “fusion complex”
Herpesvirus
Cell
gB
gH/gL
IniGal binding through aDachment factors
Receptor binding provides fusion trigger
Entry by fusion with cellular membrane
?
Fusion trigger acGvates fusion complex: gB, gH/gL
gB, gH/gL
*From Maurer et al. PNAS, 2008
*
gH/gL complex and its role(s)
• gB and gH/gL required for opGmal levels of fusion • gB -‐ the principal herpesvirus fusogen • gH/gL – In some herpesviruses has inherent fusogenic properGes
– Important for resoluGon of hemifusion intermediate
– Implicated in regulaGon of fusion acGvity of gB – Structures of gH/gL from HSV-‐2, EBV and PrV solved
Comparison of HSV-‐2 and EBV gH/gL with PrV gHC
1Chowdary TK, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, Heldwein EE. Nat Struct Mol Biol. (2010); 17:882-‐8. 2Backovic M, DuBois RM, Cockburn JJ, Sharff AJ, Vaney MC, Granzow H, Klupp BG, Bricogne G, MeLenleiter TC, Rey FA. Proc Natl Acad Sci U S A. (2010);107:22635-‐40 3Matsuura H, Kirschner AN, Longnecker R, Jardetzky TS. Proc Natl Aca Sci U S A. (2010);107:22641-‐6.
1 2 3
• The same domain organizaGon despite low sequence conservaGon • EnGrely novel fold • gL co-‐folds with the N-‐terminal, membrane distal, region of gH • gH funcGon sGll not understood
Few words about CMV gH/gL
• CMV glycoproteins not structurally characterized
• CMV gH/gL forms complexes • With other envelope proteins (gO, UL128-‐130-‐131A)
• With cellular integrin receptors • With neutralizing anGbodies
• Goal – express and characterize biochemically and structurally CMV
gH/gL and its complexes
CMV gH/gL expression in insect cells
27 718 Strep2
Full-‐length gH 27 743 718
TM ectodomain tail
gH ectodomain
31 278 gL
S2 insect cells
BiP signal pepGde
Protein of interest (gH or gL)
EK cleavage site Double strep tag
Stop codon
pT350 plasmid for expression in S2 Drosophila cells
2gH/2gL (≈224kDa)
MW (kDa)
No. of Cys
N-‐glyc. sites
gH ectodomain 84 12 6
gL 28 6 1
gH/gL 112 18 7
CMV gH/gL expression in insect cells ctd. gH and gL are bound by disulfide bond(s)
• Affinity streptacGn purificaGon
• Size exclusion chromatography
• >5mg of pure protein / 1L of S2 cell culture gH/gL (≈112kDa) gH/2gL (≈140kDa)
250 130 100 70
55
35
25
DTT + -‐
gH
gL
2gH/2gL – homodimer (of gH/gL) gH/gL -‐ heterodimer
Size exclusion separaGon of CMV gH/gL oligomers Homodimers can be separated from other species
Abs 280nm
26 30 38 50
gH/gL
gH/2gL
2gH/2gL
26 30 38 50
Which Cys residues in gH and gL are mediaGng formaGon of higher molecular weight species?
• Cys in CMV gH (12) and gL (6) • 4 in the N-‐terminal half of gH – not conserved • 8 in the C-‐terminal half of gH – conserved and paired (4 disulfide bridges) • 6 in gL – not conserved
gH ectodomain 27 718
Strep2
31 278 gL
• gL binds to the N-‐terminal end of gH • Likely candidates for unpaired Cys: N-‐terminal Cys of gH and
Cys in gL
EM analysis of CMV gH/gL species by negaGve staining Homodimers are held by Cys in gL or N-‐terminus of gH
gH/gL
gH/2gL
2gH/2gL
26 30 38 50 Fr. # Popula;on 1
(%) Popula;on 2
(%)
26 70 13
30 58 24
38 52 39
50 3 72
2gH/2gL gH/gL and gH/2gL
PopulaGon 1 PopulaGon 2 HSV-‐2 gH/gL
≈120Å
gL
gH
gL
C
C
N N
Neutralizing MSL109 mAb binds to CMV gH/gL Binding site points away from gL and gH N-‐terminus
2gH/2gL + 2 Fabs gH/gL and gH/2gL + Fab 2gH/2gL gH/gL and gH/2gL
• MSL109 mAb neutralizes virus • Tested in clinical trials • Binds to gH/gL • Localize the epitope on gH/gL • Use it as a tool to separate gH/gL
gL
VersaGlity of CMV gL Cys144 CMV gH/gL expressed in mammalian cells lacks gH/2gL form
27 718 Strep2
31 278 gL
gH ectodomain
144
gH/gL gL (gH/2gL) ?
Is gL Cys144 also mediaGng formaGon of gH/2gL? Simple gH/gLC144S heterodimers obtained in S2 cells
gH/gLC144S
gH
DTT -‐ +
250
130
92
Conclusions • Recombinant gH/gL expressed in insect cells exists as a mixture of 3 forms:
• 2gH/2gL (homodimers) • gH/2gL • gH/gL (heterodimer)
• CMV gH and gL are disulfide linked in all 3 forms • The shape of CMV gH/gL resembles HSV-‐2 gH/gL • CMV homodimers are formed through head-‐to-‐head associaGon of 2 gH/
gL molecules • Unpaired Cys in the N-‐terminus of gH or in gL mediate formaGon of
homodimers • MSL109 Fab binds close to the C-‐terminal end of gH (away from gL) • Cys144 in gL is responsible for formaGon of CMV gH/gL homodimers and
gH/2gL species • Always consider different expression hosts
Acknowledgements
Structural Virology Unit at Pasteur Ins;tute Felix Rey
University of Washington, SeaDle, USA David Veesler
