16th Workshop

"Cell Biology of Viral Infections"

of the Society for Virology (GfV)

"The Ins and Outs of Virus Infection"

Kloster Schntal

Nov 8th until Nov 10th 2017

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Cover shows infection of murine cells by Rift Valley fever virus. Nuclei appear in blue, the viral nucleoprotein in red, and the viral non-structural protein NSs in green. Photo courtesy of Dr. Psylvia Lger, Lozach lab.

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We thank the following organizations and companies for the financial support of our workshop:

Chica and Heinz Schaller (CHS) Foundation Heidelberg, Germany http://www.chs-stiftung.de

Gesellschaft fr Virologie e.V. Ulm, Germany http://www.g-f-v.org

Reblikon GmbH Schriescheim, Germany

Becton Dickinson http://www.bd.com/de

Viruses, MDPI http://www.mdpi.com/journal/viruses

Deutsche Gesellschaft fr Zellbiologie Heidelberg, Germany http://zellbiologie.de/

http://www.chs-stiftung.de/http://www.g-f-v.org/http://www.bd.com/dehttp://www.mdpi.com/journal/viruseshttp://zellbiologie.de/

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Workshop Program

Day 1 (Wednesday, November 8, 2017)

10:30 Bus Departure from Heidelberg

12:00 13:15 Welcome Lunch

13:15 13:30 Opening and Welcome Remarks Gisa Gerold and Pierre-Yves Lozach

13:30 14:30 Keynote Lecture Cell biology of influenza virus uncoating Prof. Ari Helenius ETH Zurich, Switzerland

14:30 17:20 Workshop Session Virus Entry and Intracellular Trafficking

Oral 1. Pia Brinkert, University of Mnster HPV16 endocytosis depends on BAR domain proteins and branched actin

polymerization

Oral 2. Lisa Lasswitz, Twincore, Hannover The role of CAPN5 and CBLB in HCV entry

Oral 3. Joo Miguel Freire, Institut Pasteur, Paris, France Distinct pH and endosome compartment for yellow fever wt and vaccine

strain 17D viruses revealed by single particle fluorescence microscopy

15:30 16:00 Coffee Break

Oral 4. Iris Medits, Medical University of Vienna Flavivirus E protein stem interactions in virus entry

Oral 5. Melina Vallbracht, Friedrich-Loeffler-Institut, Riems Analysis of minimal requirements for herpesvirus mediated membrane

fusion

Oral 6. Holda Anagho, University Hospital Heidelberg Mechanisms of Uukuniemi virus fusion

Oral 7. Floriane Lagadec, University of Bordeaux, France Direct detection of incoming and replicating adenoviral genomes in living cells

17:20 17:30 Short Break

17:30 18:15 Special Lecture Scientific editing Open access publishing in Virology Dr Sarai Rodrguez Navarro MDPI, Barcelona, Spain

18:30 Social Event, dinner

19:45 Group Picture and Wine Tasting in the Cellar

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Day 2 (Thursday, November 9, 2017)

7:30 9:00 Breakfast

09:00 10:00 Keynote Lecture New twists on enveloped virus entry and exit

Prof. Margaret Kielian Albert Einstein College of Medicine, New-York, USA

10:00 12:30 Workshop Session Virus Replication and Egress

Oral 8. Nicole Christin Bilz, University of Leipzig Rubella virus replication in induced pluripotent stem cells and in germ layer

cells

Oral 9. Christin Mller, Justus Liebig University Giessen Inhibition of cytosolic phospholipase A2alpha impairs coronavirus

replication by interfering with virus-induced replicative organelle formation

10:40 11:10 Coffee Break

Oral 10. Sebastian Joecks, Twincore, Hannover Pathways of intracellular lipoprotein recruitment during hepatitis C virus

assembly and release

Oral 11. Jrg P.F. Nesch, DKFZ Heidelberg Cell-to-cell transfer of parvovirus progeny through exocytic vesicles

Oral 12. Olaf Isken, University of Luebeck Identification of NS3 surface residues critical for HCV replicase assembly

or virion morphogenesis

Oral 13. Annica Flemming, University Hospital Heidelberg Design of a FRET-based sensor to monitor HIV-1 protease activity during

viral assembly

12:30 13:30 Lunch

13:30 14:30 Kloster Schntal Tour

14:30 16:10 Workshop Session Host Cell Virus Interactions

Oral 14. Anja Schbel, Leibniz Institute for Experimental Virology Interaction between the LINE1 retrotransposon and the hepatitis C virus

Oral 15. Claudia Claus, University of Leipzig The bioenergetics profile of rubella virus-infected epithelial and endothelial

cells

Oral 16. Magalie Mazelier, University of Heidelberg The tick-borne virus model Uukuniemi

Oral 17. Ana Hickford Martnez, Hannover Medical School Novel approaches to identify cytosolic host proteins interacting with the

capsids of herpes simplex type 1

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Oral 18. F. J. Zapatero Belinchon, Hannover Medical School Resistance of the non-lymphocytic cell line SH-SY5Y to filoviral cell entry is likely due to a currently unknown host factor

16:10 18:00 Poster Session (coffee will be served)

18:00 19:00 Keynote Lecture The importins of herpes simplex virus replication Prof. Beate Sodeik

Hannover Medical School, Germany

19:30 Workshop Dinner

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Day 3 (Friday, November 10, 2017)

7:30 9:00 Breakfast

09:00-10:00 Keynote Lecture Virus-activating host cell proteases: determinants of cell entry and

pathogenesis, targets for intervention Prof. Stefan Phlmann

Leibniz Institute for Primate Research, Gttingen, Germany

10:00 12:30 Workshop Session Antiviral Response and Virus control

Oral 19. Laura Soria-Martinez, University of Mnster Towards the role of glycosaminoglycan structure for HPV binding and

infection

Oral 20. Thomas Hoenen, Friedrich-Loeffler-Institut A genome-wide siRNA screen identifies a druggable host pathway as

essential for the life cycle Ebola virus

10:40 11:10 Coffee Break

Oral 21. Susann Kummer, University Hospital Heidelberg Influenza A virus infection triggers IFITM3 clustering in human lung cells

Oral 22. Allison Groseth, Friedrich-Loeffler-Institut Lifecycle modelling systems support inosine monophosphate

dehydrogenase (IMPDH) as a pro-viral factor for efficient New World arenavirus RNA synthesis

Oral 23. Venkat Raman Ramnarayan, Jacobs University Bremen

The p24 family protein TMED10/Tmp21/p241 anchors m152/gp40 in the endoplasmic reticulum to abolish MHC class I surface expression

Oral 24. Ivana Kutle, Hannover Medical School The mouse cytomegalovirus protein M25 a key player in cytomegalovirus

driven cell rounding manipulates the activity of the p53 protein in infected cells

12:30 12:40 Closing Remarks Gisa Gerold and Pierre-Yves Lozach

12:40 14:00 Lunch

14:00 Bus departure to Heidelberg

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Oral presentations

Oral 1. HPV16 endocytosis depends on BAR domain proteins and branched actin polymerization

Pia Brinkert1, Lena Khling1, Carina Bannach1, Lilo Greune2, Alexander Schmidt2,3, Theresia Stradal4, Mario Schelhaas1,3

1 Section Cellular Virology, Institutes of Virology and Medical Biochemistry, ZMBE, University of Mnster, Mnster, Germany 2 Institute of Infectiology, ZMBE, University of Mnster, Mnster, Germany 3 Cluster of Excellence EXC1003, Cells in Motion, CiM, Mnster, Germany 4 Section Cell Biology, Helmholtz Centre for Infection Research, Braunschweig

The majority of animal viruses hijack one of the various endocytic pathways to gain access to host cells. Human papillomavirus type 16 (HPV16), which is the major causative agent for cervical cancer, exploits a novel endocytic pathway for cellular uptake. This pathway shares many similarities with macropinocytosis, but differs in endocytic vacuole formation as evidenced by e.g. different vesicle morphology. We investigated the requirements for HPV16 endocytosis and aimed to understand its regulation in comparison to macropinocytosis. Using live cell imaging, electron microscopy and cellular perturbations during infection, we compared HPV16 endocytosis to the macropinocytic uptake of vaccinia virus. The membrane bending BAR domain proteins SNX2 and ASAP1 were found to regulate HPV16 endocytosis. In contrast, the major BAR domain protein involved in macropinocytic cup closure, CtBP1, was dispensable for HPV16 uptake. Moreover, vesicle scission was mediated by actin polymerization. A more detailed analysis of the regulation of actin polymerization revealed that branched filaments induced by the Arp2/3 complex were responsible for scission of HPV16 vesicles. Interestingly, in contrast to vaccinia virus uptake. the recruitment of the Arp2/3 complex did not occur by actin nucleation promoting factors typically found at the plasma membrane.

Oral 2. The role of CAPN5 and CBLB in HCV entry

Lisa Lasswitz, Janina Bruening, Pia Banse, Sina Kahl, Arnaud Carpentier, Florian Vondran, Lars Kaderali, Thomas Pietschmann , Felix Meissner and Gisa Gerold

The hepatitis C virus (HCV) causes a global health burden affecting nearly 71 million people worldwide. HCV enters human hepatocytes through a multistep process involving four essential host factors, one of them being the cell surface tetraspanin CD81. In an in-depth analysis of the CD81-interactome in human hepatocytes using high-resolution mass spectrometry based proteomics, we identified the CD81-interactors calpain-5 (CAPN5) and the E3 ubiquitin-protein ligase Casitas B-lineage lymphoma proto-oncogene b (CBLB) as specific entry factors for HCV. Cells with a CRISPR/Cas9-induced knock out of either of the enzymes show significantly reduced infection with all seven HCV genotypes. However, how these two proteins fulfil their role is not clear so far. Therefore, our project aims to further elucidate the function of CAPN5 and CBLB in HCV infection. We hypothesized that CAPN5 and CBLB regulate HCV-receptor complex internalization through their enzymatic activity. First, we tested if surface levels of the CBLB substrate and HCV co-receptor epidermal growth factor receptor (EGFR) are altered by CBLB knock out using fluorescent antibody staining. Then, we cloned CAPN5- and CBLB-variants containing a guide RNA binding site mutation with and without an additional active site mutation. We then transduced the knock out cell lines with pseudoparticles (pp`s) encoding these CAPN5- or CBLB-variants. To see if the overexpression of both protein variants can boost HCV infection, we also transduced control cells with these pp`s. In parallel, we tested a specific CBLB-inhibitor in a human hepatoma cell line as well as in primary human hepatocytes. Further on to confirm the role of CAPN5 and CBLB in stem cell-derived hepatocyte-like cells, we silenced the two proteins using siRNA knock down. We observed that EGFR surface expression levels are not affected by CAPN5 and CBLB knock out. Moreover, CBLB wild-type and enzymatically inactive mutant can both rescue the CBLB knock out, while the CAPN5 wild-type seems to rescue the knock out and the enzymatically inactive mutant rescues less efficiently. In line with this data, the CBLB inhibitor does not seem to inhibit

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HCV infection at an intermediate concentration in hepatoma cells as well as in primary human hepatocytes. Finally, we demonstrate in a first experiment that CAPN5 and CBLB are also required for HCV infection of stem cell-derived hepatocyte-like cells as the knock down of both proteins leads to reduced infection rates. In conclusion, we confirm a role of CAPN5 and CBLB in human hepatoma cells and stem cell-derived hepatocyte-like cells with different requirements for enzymatic activity of the two novel entry factors.

Oral 3. Distinct pH and endosome compartment for Yellow fever wt and vaccine strain 17D viruses revealed by single particle fluorescence microscopy.

Joo Miguel Freire, Eugenia Covernton, Felix Rey and Giovanna Barba Spaeth

Structural Virology Unit, Department of Virology, Institut Pasteur, Paris, France

Yellow fever (YF) virus is an enveloped RNA virus belonging to the flavivirus genus that enters the cell via receptor-mediated endocytosis. The YF envelope protein (E), which covers the surface of the virion, when exposed to the acidic pH of the endosome, undergoes structural rearrangements driving endosome-virion membrane fusion and release of the viral RNA. The attenuated strain YF 17D is currently used has a vaccine. Interestingly, 12 out of the 32 aa mutations of YF 17D are clustered in the E protein, most of them localized in regions responsible for the pH-induced conformational changes during viral fusion. Such observation suggests that cellular fusion for the two strains may occur at different pH, and eventually within different endosome compartments. Thus, this work has the goal to determine the endosome pH at which each YF virus fuse with the host membrane using single particle confocal microscopy. We compared the endocytic pathway of YF 17D and YF Asibi during cellular infection. For this purpose we monitored the cellular entry of YF viruses together with a pH sensitive probe (pHrodo) that enters the cell at the same time as the virus and allows the quantification of the pH at each time point during virus entry. Single particle fluorescence microscopy of YF DiO-labeled viruses allowed us to identify viral fusion events and further quantify the pH at which the fusion occurs by co-localization. Our results (confocal microscopy and flow cytometry) showed that YF 17D membrane fusion occurs in a more acidic pH than for YF Asibi. Moreover, the quantification of the pH at each endocytosis compartment (early, late and lysosome) suggests that YF 17D fuses in endosomes with late endosome pH whereas YF Asibi fuses in early endosomes. Co-localization studies of YF fusion events with specific endosome markers (EEA-1, Rab5, Rab7 and Rab9) supported this statement. The majority of YF 17D fusogenic particles identified were localized in Rab9 endosomes, thus in late endosomes. We also explored the fusion capacity of YF 17D and YF Asibi in vitro using liposomes that mimic each endosome compartment in their lipid composition and environment pH. The data supports that YF 17D fusion with lipid membranes occurs at very acidic pH and in the presence of late endosome-like lipid membranes. As for YF Asibi, fusion with early endosome-like membranes was observed. To conclude, we observed that YF 17D and YF Asibi viral fusion occurs at different pH and lipid environment, in different endosome compartments, late and early endosomes, respectively. Such finding will provide deep insight to understand the molecular/cellular factors that lead the host to a protective immune response against YF 17D and contribute to new designing strategies of novel vaccines.

Oral 4. Flavivirus E protein stem interactions in virus entry

Iris Medits, Franz X. Heinz, Karin Stiasny

Department of Virology, Medical University of Vienna, Vienna, Austria

INTRODUCTION: Flaviviruses enter cells by receptor-mediated endocytosis. After virus uptake, the viral envelope protein E mediates fusion of the viral and the endosomal membrane, triggered by the slightly acidic pH in endosomes. The current fusion model is based on atomic structures of truncated forms of the E protein in their dimeric pre- and trimeric post-fusion conformation. These structures lack the two transmembrane-domains and the so-called stem-region. The stem connects the ectodomain and the membrane anchor and is hypothesized to be essential for fusion by zippering along the trimer core during the conformational changes of E necessary for fusion.

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OBJECTIVES: Since stem interactions are essential for fusion and hypothesized to provide part of the energy required for this process, we wanted to gain information on these interactions as well as their role in the fusion process by a mutagenesis approach using tick-borne encephalitis virus (TBEV), a major human pathogenic flavivirus.

Materials and Methods. We introduced modifications (point mutations, deletions) into the stem of recombinant E proteins as well as an infectious clone of TBEV and analyzed their effect on E protein trimerization, trimer stability and infectivity.

RESULTS AND CONCLUSION: We identified important interaction sites between the stem and the trimer core involved in the stabilization of the post-fusion conformation. In addition, replacing conserved residues in the stem resulted in a strong reduction in the production of infectious particles. Passaging experiments allowed the identification of resuscitating mutations which led to a recovery of the viability of mutant viral particles to wildtype levels. Currently, we investigate whether the observed phenotypes are caused by defects in virus entry, assembly or both processes.

Oral 5. Analysis of minimal requirements for herpesvirus mediated membrane fusion

Melina Vallbracht, Walter Fuchs, Barbara G. Klupp, Thomas C. Mettenleiter

Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems

Membrane fusion is crucial for entry and spread of enveloped viruses. In herpesviruses this process is mediated by three conserved viral glycoproteins, (g)B and the heterodimeric gH/gL complex, plus other nonconserved species specific receptor binding proteins like alphaherpesvirus gD. Although gB carries signatures of a bona fide fusion protein, membrane fusion depends on presence of the gH/gL complex, whose role, however, remains elusive. In pseudorabies virus (PrV), an alphaherpesvirus of swine, function of gL as well as the gL-binding domain of gH can be compensated by mutations in gB, gH and/or gD. In addition, recent data indicated that in the related herpes simplex virus soluble forms of the gH/gL complex without transmembrane anchor might be sufficient to mediate fusion in combination with gB, while other results pointed to important functions of the transmembrane (TMD) and cytoplasmic domain (CD) of gH. To further identify functionally important regions in PrV gH we generated mutant proteins lacking either the CD and/or the TMD, or substituted these regions by heterologous sequences. The mutated gH-proteins were tested for their ability to mediate membrane fusion in transfection based cell-cell fusion assays and to complement gH-negative PrV. Our results demonstrate that the PrV gH CD is dispensable for function but the TMD plays a prominent role. Surprisingly, a single point mutation within the TMD was sufficient to compensate the lack of gL and the gL-binding domain of gH.

Oral 6. Mechanisms of Uukuniemi virus fusion

Holda Anagho1, Nele Villabruna1, Sven Blobner1, Pablo Guardado-Calvo2, Flix Rey2, Pierre-Yves Lozach1

1 Department of Infectious Diseases, Virology, University Hospital Heidelberg, Germany 2 Institut Pasteur, Unit de Virologie Structurale, Institut Pasteur, Paris, France

The Uukuniemi Phlebovirus (UUKV) is closely related to important emerging pathogenicviruses in the Phenuiviridae family causing severe and sometimes fatal disease in humans, animals, and plants. UUKV enters cells by fusing its membrane with host cell membranes. Previous studies have shown that Glycoprotein C (GC) of phleboviruses, which mediates fusion, have characteristics of class II fusion proteins. The ectodomain of GC is composed of three domains (DI-DIII) connected to a transmembrane anchor in the viral envelope by a stem region. The class II fusion machinery is activated by the low pH environment in endosomes encountered during entry. Interestingly, we found that when UUKV was pretreated at low pH for 5 minutes, the virus did not lose its infectivity, suggesting that some part of the fusion process is reversible. To characterize residues in GC important for UUKV fusion, we made an S914A mutation to abolish N-glycosylation of N912, hypothesized to be important in stabilizing the post-fusion GC trimer. The S914A mutation led to a 10-fold lower titer of rescue UUKV (rUUKV) compared to wild type rUUKV, but did not affect the kinetics of infection up to 48 hours, indicating that this residue alone is not critical for stabilizing the post-fusion GC trimer. Mutations were also made in the cd loop (A646W) corresponding to W821

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in the Rift Valley Fever Phlebovirus (RVFV), or the ij loop (D961K), highly conserved in phleboviruses. However, infectious rUUKV containing these mutations could not be rescued from cDNA. Moreover, no GC was detected in lysates of transfected cells after 1 or 4 days by western blot, suggesting that A646W or D961K mutant particles were not assembled. Lastly, peptides corresponding to DIII, the stem, and the C-terminal membrane permeable region (MPR) were tested for their ability to inhibit UUKV fusion at the cytoplasmic membrane. DIII alone did not inhibit fusion while peptides containing DIII, stem, and half of the MPR were most potent in inhibiting fusion. These results provide important functional studies to accompany the recently solved crystal structures of phlebovirus GC ectodomain and lay the basis for the development of new therapeutic strategies against phleboviruses.

Oral 7. Direct detection of incoming and replicating adenoviral genomes in living cells

Floriane Lagadec1, Tetsuro Komatsu1, Frank Gallardo2 and Harald Wodrich1

1 MFP CNRS UMR 5234, University of Bordeaux, France 2 NeoVirtech, Toulouse, France

Nuclear delivery of viral genomes upon entry is a prerequisite to commence a productive replication for many DNA viruses. However very little knowledge exists concerning how viral genomes are delivered into the nucleus and what happens to them after nuclear import prior to and during viral replication. This lack of information is largely due to missing experimental systems that allow the detection of individual viral genomes in the nucleus of infected cells. Recently novel systems like the labeling of viral genomes with modified nucleotides suitable for click-chemistry (e.g. EdU, EdC) have been developed permitting the detection of individual viral genomes in fixed cells. In contrast systems, which can detect individual viral genomes in living cells to gain spatio-temporal resolution are still missing. Here we report on our efforts to overcome this technical barrier for adenoviral genomes. Adenoviruses (AdV) are nuclear replicating non-enveloped DNA viruses. Adenoviral genomes are packed into viral chromatin (core) inside the entering capsid. The 36kb linear DNA molecules is compacted via ~800 copies of the histone-like core proteins VII and transported inside the partially disassembled capsid towards the nuclear pore. At the nuclear pore the capsid disassembles and the genome is released and imported into the nucleus exposing the pVII epitope e.g. amenable to antibody detection. To visualize incoming genomes in living cells we developed a first generation visualization system relying on a cellular factor, TAF-1, that binds specifically to pVII on incoming genomes. Using GFP-tagged TAF-1 we are now able to image individual incoming viral genomes in living cells. We recently also developed a novel system tagging the viral DNA itself using Anchor technology. This system is based on a proprietary sequence inserted into the viral genome, which is recognized by the dynamic recruitment of a GFP-tagged protein, thus allowing genome detection in living cells with improved signal-to-noise ratios. Moreover this system is suitable to follow viral genome replication in living cells in real time.

Oral 8. Rubella virus replication in induced pluripotent stem cells and in germ layer cells

Nicole Christin Bilz1, Denise Hbner1, Janik Bhnke1, Uwe Gerd Liebert1, Claudia Claus1

1 Institute of Virology, University of Leipzig, Leipzig, Germany

Rubella virus (RV) embryopathy is restricted to humans. Despite intensive investigations the cellular alterations leading to congenital defects after maternal RV infection during the first trimester of pregnancy are still not understood. Thus, new model systems are required to understand its underlying mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising in vitro cell culture model for the very early phase of human embryonic development from the blastocyst stage up to early gastrulation. hiPSCs were infected with the vaccine strain RA27/3 and two clinical isolates of RV, one with a low and one with a high rate of cytopathogenicity on somatic cells. All three RV strains replicated equally well in hiPSCs. However, the low cytopathogenic clinical isolate required more passages of infected hiPSC colonies to achieve a high rate of infection compared to the one with a higher rate of cytopathogenicity. All three RV strains enabled differentiation of passaged and infected hiPSCs to cells of the three germ layers. Notably, highest rate of replication was seen in mesoderm. Descendants of this germ layer develop into vascular cells. Congenital

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vascular defects are severe after maternal infection with RV. This suggests that hiPSCs are a suitable cell culture model for RV associated defects during early embryogenesis.

Oral 9. Inhibition of cytosolic phospholipase A2alpha impairs coronavirus replication by interfering with virus-induced replicative organelle formation

Christin Mller1, Martin Hardt2, Dominik Schwudke3, Benjamin W. Neuman4, Stephan Pleschka1, and John Ziebuhr1

1 Institute of Medical Virology, Justus Liebig University Giessen, 35392 Giessen, Germany 2 Imaging Unit, Biomedical Research Center, Justus Liebig University Giessen, 35392 Giessen, Germany 3 Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz Center for Medicine and Bioscience, 23845 Borstel, Germany 4 Texas A&M University, Texarkana, TX 75503, USA

Similar to other +RNA viruses, coronaviruses induce membrane rearrangements in infected cells, resulting in organelle-like virus factories that carry multi-subunit complexes that drive viral RNA synthesis. These replicative organelles (ROs) contain the viral and cellular proteins involved in viral replication/transcription and may also help to sequester viral factors from recognition by host defense mechanisms. There is increasing evidence that enzymes involved in cellular lipid metabolism have important roles in RO formation and, possibly, other steps of the coronavirus replication cycle. In this study, we assessed the role of cellular cytosolic phospholipase A2alpha (cPLA2a) activity in the replication of corona- and other RNA viruses. cPLA2a catalyzes the hydrolysis of membrane-associated glycerophospholipids at the sn-2 position, releasing a fatty acid and generating a lysophospholipid (LPL). Effects of cPLA2a-specific inhibitors on RO formation in virus-infected cells were studied by confocal laser-scanning and transmission electron microscopy, while effects on viral replication were studied by Northern and Western blotting and virus titration. In human coronavirus 229E (HCoV-229E)-infected Huh-7 cells, inhibition of cellular cPLA2a activity was found to impair viral protein and RNA accumulation and resulted in reduced numbers of ROs. Furthermore, inhibition of cPLA2a activity resulted in reduced LPL levels, suggesting an involvement of LPLs in producing the membranous structures required for coronavirus replication. This is further supported by lipidome analyses of HCoV-229E-infected (compared to mock-infected) cells, which revealed an increase of LPLs in virus-infected cells. This increase is suppressed in the presence of cPLA2a inhibitor. Finally, we were able to confirm that inhibition of cPLA2a activity affects the replication of several other +RNA viruses known to induce intracellular membrane rearrangements, such as MERS-CoV and Semliki forest virus, whereas poliovirus, human rhinovirus 1A, and vaccinia virus replication was not affected. Taken together, the data from this and a previous study provide strong evidence to suggest that cellular cPLA2a activity has important roles at different steps of the replication cycle of +RNA viruses from different families.

Oral 10. Pathways of intracellular lipoprotein recruitment during hepatitis C virus assembly and release

Sebastian Joecks1, Annika Frauenstein2, Janina Brning1, Gisa Gerold1, Gabrielle Vieyres1, Felix Meissner2 and Thomas Pietschmann1

1 Institute for Experimental Virology, Twincore, Feodor-Lynen-Strae 7, 30625 Hannover, Germany 2 Department for Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Planegg

Infections with the hepatitis C virus (HCV) are a global health burden affecting around 71 mio people worldwide. Over decades chronic infections can lead to end-stage liver diseases (ESLD), being to date the first indication for liver transplantation in industrialized countries. HCV is a small enveloped, positive-sense, single-stranded RNA virus that belongs to the family of Flaviviridae within the genus of hepacivirus. The viral replication cycle is known to be inseparably connected to the host cell lipid metabolism. Beside lipoprotein-mediated entry of the virus, several host cell factors are hijacked by the virus to facilitate viral assembly. At later assembly steps interactions with apolipoproteins, like ApoE, and other components of lipoprotein pathways occure. This ultimately leads to the release of so-called lipo-viral particles (LVPs) that resemble particles of the host cellular VLDL-pathways. Independently of these findings, the precise composition of

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HCV assembly complexes and the machineries coordinating intracellular decoration of HCV with lipoproteins are incompletely understood. Our project aims to identify viral and host cellular proteins and pathways contributing to HCV assembly and lipoprotein recruitment. For this we generated and characterized virus variants carrying epitope-tags at proteins known to be important for assembly. Further on we made use of a human hepatoma cell line, in which an important host factor for HCV assembly is epitope-tagged, too. In the context of HCV infection we conducted and established an immunoprecipitation protocol for purification of these factors and analyzed them by label-free quantification mass-spectrometry (AP-LFQ-MS/MS). Several important factors were identified by this approach, mainly being connected to host cellular ER-pathways and proteins. Newly identified proteins are validated by co-precipitation with the respective epitope-tagged proteins. Further we will show the influence of these factors on virus assembly by siRNA knockdown, live-cell imaging and confocal microscopy to visualize the interactions between identified proteins and the viral replication cycle. Besides wild-type virus, we generated and characterized viral mutants arrested at distinct steps of HCV morphogenesis, allowing identification of interactomic changes during the assembly process. Further MS-analysis will be performed with these viruses. The identification of proteins and pathways contributing to efficient HCV assembly and release will provide profound knowledge about how virus infections can alter/ hijack the host cells metabolism for their own purpose.

Oral 11. Cell-to-Cell Transfer of Parvovirus Progeny Particles through Exocytic Vesicles

Clemens Bretscher, Tamara Steinbach, Sverine Br, Jean Rommelaere, Jrg P.F. Nesch

Rodent parvoviruses are small icosahedric, non-enveloped particles with a 5.1 kb single-stranded DNA as a genome. Due to their intrinsic oncotropism and oncosuppressive properties they are considered excellent candidates for a virotherapy of cancer. Efficient cancer therapy using self-propagating oncolytic viruses is based on their capability to infect cancer cells and spread through the diseased tissue, thereby inducing an anti-tumor immune response. Focusing on late stages of the virus cycle it was shown that progeny PV particles are actively transported from the nucleus to the cell periphery where mature virions are released. This is a regulated process involving the activity of viral and cellular proteins. In fact, we could show that after release from the nucleus, with the aid of gelsolin and the ERM family proteins moesin/radixin, progeny particle become engulfed into COPII-vesicles in the perinuclear area and are guided by Rdx, rab1 and rab11 through ER, Golgi and (recycling) endosomes or multivesicular bodies to the plasma membrane. Although non-enveloped lytic viruses like PVs are thought to be released through a burst of lysis, we also observed shedding of progeny particles prior to disintegration of infected cells. These viruses appear to be engulfed in vesicles, opening up new avenues for cell-to-cell spread of PVs. Current research tries to characterize extracellular PV-containing vesicles and to unravel mechanisms and pathways involved in release and spreading.

Oral 12. Identification of NS3 surface residues critical for HCV replicase assembly or virion morphogenesis

Olaf Isken, Hella Schwanke, Corinna Erfurth and Norbert Tautz

Institute of Virology and Cell Biology, University of Luebeck, 23562 Luebeck, Germany

The assembly of hepatitis C virus (HCV) replicase complexes (RCs) is a highly regulated process that involves many viral and cellular factors. Attempts to understand the assembly of the HCV RC have been challenging due to limitations in our understanding of the protein-protein interactions required. At the center of the regulation of RC formation is NS3, a multifunctional protein with roles in polyprotein processing, RNA replication and virion morphogenesis. Recently, we demonstrated that a hydrophobic patch on the surface of the NS3 protease domain is critical for multiple steps during the temporal regulation of RC assembly. At first this NS3 surface area promotes NS2 protease activity to allow for efficient NS2-NS3 cleavage, a prerequisite for RC formation. Only after NS2-NS3 cleavage has occurred, this specific NS3 surface area becomes engaged in its second function, namely in protein-protein interactions leading to RC assembly.

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In the present study, we identified novel surface residues critical for functional RC assembly and RNA replication within this multifunctional NS3 surface. Interestingly, some of these determinants reside in the NS3 helicase domain close to the protease domain and form together with our recently identified surface patch on the protease domain a contiguous surface area on NS3 that most likely serves as the initial interaction platform in RC assembly. NS3 is also involved in virion morphogenesis. Interestingly, we identified one residue within the newly characterized NS3 surface region on the helicase domain, whose mutation was detrimental for virion production without affecting RNA replication / RC maturation. This illustrates the multipurpose use of protein surfaces to assemble functionally different multiprotein complexes. Taken together, our present data set is providing a basis to further dissect the formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.

Oral 13. Design of a FRET-based sensor to monitor HIV-1 protease activity during viral assembly

Annica Flemming1, Vibor Laketa1, Ulrike Engel2, Hans-Georg Krusslich1 and Barbara Mller1

1 University Hospital Heidelberg, Department of Infectious Diseases, Virology 2 Heidelberg University, Nikon Imaging Center and Centre for Organismal Studies

Human immunodeficiency virus (HIV-1) forms new viral particles in infected cells via assembly at the plasma membrane. The main structural polyprotein Gag assembles at the membrane and recruits other viral components and cellular factors required for release. To prepare the virus for infecting a new target cell, the polyproteins Gag and GagProPol have to be cleaved by the viral protease (PR), followed by a dramatic structural rearrangement within the particle. This process is called maturation. The correct timing and order of proteolytic cleavage is crucial for the formation of infectious viral particles. However, the trigger for PR activation and the time course of proteolytic maturation are currently unclear. Therefore, we designed a Frster resonance energy transfer (FRET)-based sensor to monitor HIV-1 PR processing during the viral assembly process. For this, the fluorescent proteins Clover and mScarlet were linked via a PR cleavage site and coupled to the viral protein r (Vpr) to mediate incorporation into the viral particle. Clover serves as a donor fluorophore for mScarlet; proteolytic separation of the fluorophores should result in a decrease of FRET signal. In purified virus-like particles (VLP) we indeed observed a 30-40% decrease of the FRET signal when comparing wild-type and PR deficient particles. Acceptor bleaching experiments were conducted in virus producing cells to assess the maximal expected FRET signal change at HIV-1 assembly sites for total internal reflection fluorescence (TIRF) microscopy. Therefore, a PR deficient virus was co-expressed with the Clover-mScarlet.Vpr sensor. Bleaching of mScarlet resulted in 25-30% signal increase of Clover. Late HIV(wt) assembly sites at the ventral plasma membrane displayed a threefold lower FRET signal, compared to assemblies of HIV(PR-), indicating that we can detect differences in proteolytic activity by TIRF microscopy of living cells. We are currently studying the change of FRET signal during virus particle formation by live-cell imaging of virus producing cells. This should allow us to analyze the process to gain a better understanding of dynamics of this crucial step in the virus life cycle.

Oral 14. Interaction between the LINE1 retrotransposon and the hepatitis C virus

Anja Schbel1, Gerald Schumann2, Eva Herker1

1 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 2 Paul-Ehrlich-Institute, Langen, Germany

Knowledge of interplay between the hepatitis C virus (HCV) and the host is essential to understanding the establishment of chronic HCV infection. Until now, several cellular antiviral as well as proviral factors have been described that play a role at different steps of the viral life cycle. Using a proteomic approach, we identified the Long-Interspersed-Nuclear-Element1 (LINE1)-ORF1 protein (L1ORF1p) to be enriched in lipid droplet (LD) fractions of HCV-infected hepatoma cells. LINE1 belongs to the family of retrotransposons and is the only element that is still active in the human genome. It encodes for three proteins: the recently described ORF0 in antisense orientation in the 5 UTR, the RNA binding protein ORF1p and ORF2p. ORF2p encodes for a reverse transcriptase as well as an endonuclease. Integration of the LINE1 element has been linked to several single gene diseases, but little is known in context of viral infection. Western blot analysis

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revealed an overall increase of the L1ORF1p protein levels in HCV-infected Huh7.5 cells and confirmed the enrichment of L1ORF1p in lipid-rich fractions. Immunofluorescence microscopy of HCV-infected Huh7 cells overexpressing ORF1p-HA verified close proximity of ORF1p to LDs. HCV replication alone did not change protein levels, whereas preliminary data shows that the overexpression of HCV core induced the enrichment of L1ORF1p in LD fractions. This suggests a link between core protein trafficking to LDs and L1ORF1p recruitment. Co-immunoprecipitation of overexpressed HA-tagged L1ORF1p did not reveal a direct interaction of L1ORF1p and core or NS5A, which is also found on the LD surface of infected cells. Instead, we observed binding of L1ORF1p to the HCV RNA, making it a putative template for reverse transcription by L1ORF2p. Using an eGFP-based reporter assay, we analyzed the frequency of retrotransposition in HCV-infected versus uninfected cells. We observed less LINE1 retrotransposition events in HCV-infected cells compared to the nave control, which might be due the activation of antiviral signaling in the infected cells. Thus, HCV infection impacts retrotransposition; if, vice versa, retroelements affect HCV replication is currently under investigation.

Oral 15. The bioenergetic profile of rubella virus-infected epithelial and endothelial cells

Kristin Jahn1*, Nicole Christin Bilz2, Anja Ldtke2, Denise Hbner2, Judith Hbschen3, Annette Mankertz4, Uwe Gerd Liebert2, Claudia Claus2

equally contributed 1 Institute of Virology and Faculty of Biology, Pharmacology and Psychology, University of Leipzig * Current address: Institute of Biology and SIKT, University of Leipzig, Leipzig, Germany 2 Institute of Virology, University of Leipzig, Leipzig, Germany 3 WHO European Regional Reference Laboratory for Measles and Rubella, Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette, Grand-Duchy of Luxembourg 4 WHO European Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute, Berlin, Germany

The regulation of cellular metabolic pathways is flexible and allows for cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. Known for its close association with mitochondria, rubella virus (RV) is a suitable candidate for assessment of virus-induced metabolic alterations. The analysis of the respiratory (based on oxygen consumption rate, OCR) and glycolytic (based on extracellular acidification rate, ECAR) capacity enabled generation of a bioenergetic profile of RV-infected cells. Irrespective of their metabolic background, RV shifts epithelial (A549 and Vero) and endothelial (HUVEC) cells to a higher energetic level. There was a strain-specific correlation between the ability to increase metabolic activity and viral protein synthesis, but this was not connected with a certain genotype. The data presented reveal that viral influence on cellular metabolism needs to be recognized as an important contributing factor in virus-host interaction and coevolution. The capacity of RV to induce metabolic alterations appears to have evolved differently among RV strains during natural infection.

Oral 16. The tick-borne virus model Uukuniemi

Magalie Mazelier, Britta Brgger, Vojetch Zila, Hans-Georg Krusslich and Pierre-Yves Lozach

Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany

Novel tick-borne pathogenic phleboviruses in the Bunyaviridae family have lately emerged through distinct continents, all closely related to Uukuniemi virus (UUKV). To recapitulate tick-mammal switch in vitro, we established a reverse genetics system to rescue UUKV (rUUKV) from plasmid DNAs. Our recent results show that UUKV derived from tick vector cells presents original structural and glycosylation characteristics. The amount of structural protein per infectious particle units appear to be lower in rUUKV particles produced from tick cells than in those derived from mammalian cells, suggesting that arthropod vector-derived viruses are more infectious. When rUUKV produced in tick or mammalian cells is analyzed by mass spectrometry for the lipid composition, our results suggest that tick cell-derived rUUKV has a different feature than that of mammalian-derived viruses, which harbors atypical sphingolipids. Furthermore, electron microscopy and tomography of rUUKV seem to show smaller particles for the tick cell-derived viruses. Altogether, our results indicate that tick cell-derived viruses have a unique fingerprint that possibly make them more infectious. This study also highlights the importance to work with viruses

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originating from arthropod vector cells in investigation into the cell biology of arbovirus transmission and entry.

Oral 17. Novel approaches to identify cytosolic host proteins interacting with the capsids of herpes simplex type 1

Ana Hickford Martnez1, Todd Greco2, Fenja Anderson1, Daniela Kieneke1, Manutea Serrero1, Anne Binz1, Ute Prank1, Rudi Bauerfeind3, Ileana Cristea2, and Beate Sodeik1

1 Institute of Virology, Hannover Medical School, Hannover, Germany 2 Department of Molecular Biology, Princeton University, New Jersey, USA 3 Research Core Facility for Laser Microscopy, Hannover Medical School, Hannover, Germany

Herpes simplex virus has a double stranded DNA genome of 120 to 230 kb which is encased in an icosahedral capsid 125 nm in diameter, covered by a tegument consisting of about 30 different viral proteins and a glycoprotein-containing envelope. Cytosolic capsids interact with various host proteins during nuclear targeting after cell entry and during assembly prior to secondary envelopment. Herpes simplex virus type 1 (HSV1) productively infects epithelial cells and keratinocytes of mucosal membranes and skin, establishes persistent infection in sensory neurons which innervate these regions, and replicates to a limited extent in macrophages and dendritic cells. We have developed biochemical cell-free assays to reconstitute several molecular events during cytosolic capsid passage. HSV1 capsids of defined protein composition were isolated from the nuclei of infected cells or from extracellular virions and incubated with cytosolic extracts derived from murine brains that are enriched for neurons. Capsids and associated host factors were purified through sucrose cushions or by immunoisolation, and analysed by quantitative TMT (tandem mass tag) mass spectrometry, immunoblotting and electron microscopy. Both capsid types contained identical amounts of major and minor capsid proteins, but the viral capsids were coated predominantly by inner and also some outer tegument proteins, whereas the nuclear capsids were tegument-free. Furthermore, we could identify microtubule motors and constituents of multivesicular bodies that preferentially associated with viral capsids. Such tegumented capsids are translocated along microtubules in vitro and in cells, and may rely on factors involved in vesicles formation for their secondary envelopment in the cytoplasm. With these new assays that reconstitute key events of the herpes simplex virus cycle, we plan to identify novel cell-type specific host factors that may counteract or promote virion formation, and in the future to identify novel host factors important for infection of neurons and immune cells.

Oral 18. Resistance of the non-lymphocytic cell line SH-SY5Y to filoviral cell entry is likely due to a currently unknown host factor

F. J. Zapatero Belinchon1,2, E. Dietzel3, O. Dolnik3, K. Dhner4, R. Costa1,2,5, B. Hertel1, N. Atenchong1,2, M. P. Manns1, S. Ciesek5, B. Sodeik4, S. Becker3, T. von Hahn1,2

1 Hannover Medical School, Department of Gastroenterology, Hepatology, Enterology, Hanover, Germany 2 Hannover Medical School, Institute of Molecular Biology, Hanover, Germany 3 Philipps University Marburg, Institute of Virology, Marburg, Germany 4 Hannover Medical School, Institute of Virology, Hanover, Germany 5 Essen University Hospital, Institute of Virology, Essen, Germany

Ebola virus disease (EVD) was the cause of the infamous 2013-2016 West African outbreak. Although in early stages of infection macrophages, monocytes, and dendritic cells are the primary target, Filoviruses have a wide cell tropism. They are able to infect virtually any cell type with the exemption of cell lines of lymphocytic origin (Wool-Levis and Bates. 1998) and later confirmed in vivo (Geisbert et al. 2000). Moreover, Dube et al. demonstrated that EBOV refractory suspension cells (293F and lymphocytic cell lines) contain an intracellular pool of putative EBOV RBR binding partners that become translocated to the surface upon cell attachment. An unbiased lentiviral glycoprotein (GP)-driven Filovirus susceptibility cell screening identified SH-SY5Y, a neuroblastic adherent non-lymphocytic cell line, as a novel cell line refractory to Filovirus entry. These results were later validated by rVSV-EBOV-GP and authentic Filovirus infection. Here, we set out to characterize the Filovirus resistance nature of SH-SY5Y. Firstly, clustering analysis of gene array of a panel of cell lines failed to correlate Filovirus entry factor gene expression with

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susceptibility/resistance. Next, protein expression and functionality studies suggested that Filovirus entry surface factors (ITGVA, ITGB1, FOLR1, Axl, TIM-1) were heterogeneously expressed among resistant and susceptible cell lines, and intracellular factors (NPC1, TPC1/2, CTSL, CSTB) were present in most of the cell lines and functional regardless the cells susceptibility phenotype. In order to rule out the possibility of a dominant restriction factor as the cause of resistance, cell fusion assays were conducted. Strikingly, cell fusion between the resistant cell line SH-SY5Y and the permissive cell line 293T rendered heterokaryons susceptible to EBOV and MARV pseudoparticles. Importantly, lentiviral particles lacking envelope GP failed to transduce heterokaryons, suggesting that lentiviral entry is GP-specific. In conclusion, all these data suggest that expression of previously reported entry factors are not sufficient to explain Filovirus resistance on SH-SY5Y and yet to be discovered essential/s entry factor likely exist factors (NPC1, TPC1/2, CTSL, CSTB) were present in most of the cell lines and functional regardless the cells susceptibility phenotype. In order to rule out the possibility of a dominant restriction factor as the cause of resistance, cell fusion assays were conducted. Strikingly, cell fusion between the resistant cell line SH-SY5Y and the permissive cell line 293T rendered heterokaryons susceptible to EBOV and MARV pseudoparticles. Importantly, lentiviral particles lacking envelope GP failed to transduce heterokaryons, suggesting that lentiviral entry is GP-specific. In conclusion, all these data suggest that expression of previously reported entry factors are not sufficient to explain Filovirus resistance on SH-SY5Y and yet to be discovered essential/s entry factor likely exist.

Oral 19. Towards the role of glycosaminoglycan structure for HPV binding and infection

Laura Soria-Martinez1,2,3, Laura Hartmann4, Mario Schelhaas1,2,3

1 Section Cellular Virology, Institutes of Virology and Medical Biochemistry, ZMBE, University of Mnster, Mnster, Germany 2 Cluster of Excellence EXC1003, Cells in Motion, CiM, Mnster, Germany 3 DFG Research Group FOR 2327 ViroCarb: Glycans Controlling Non-Enveloped Virus Infections 4 Institute of Organic and Macromolecular Chemistry, Heinrich-Heine University of Dsseldorf, Dsseldorf, Germany

Human papillomaviruses (HPV) are small, non-enveloped DNA tumor viruses. They rely on glycosaminoglycans to attach to host cells. Specifically, HPVs require heparan sulfate proteoglycans (HSPG) for initial binding to the basal cells in the skin and/or mucosa. Upon binding to HSPGs, initial conformational changes in the HPV-16 virion occur, which facilitate further essential structural processing of the capsid prior to endocytosis. Interestingly, the structural change induced by heparan sulfates (HS) appears to depend on length, charge and sulfation patterning of the HS glycan. Here, we aimed to determine the required HS structure for the binding and subsequent structural activation of the virus. Our results indicated that an HS consisting of more than 20 saccharide units was required for structural activation. Both N- and 6-O sulfations were important for HPV infection, but only N-sulfation was crucial for the induction of structural activation. Interestingly, 2-O sulfation had a detrimental role for HPV infection. Therefore, we propose that individual HS sulfations participate in different manners in regards to binding and/or structural activation of HPV-16, and therefore, specific sulfation patterns are required to facilitate HPV-16 infection. In a first attempt to exploit this information for anti-viral measures, synthetic, so-called (precision)-glycomacromolecules were used to block binding and structural activation, the results of which will also be discussed.

Oral 20. A genome-wide siRNA screen identifies a druggable host pathway as essential for the life cycle of Ebola virus

Scott Martin1, Abhilash Chiramel2, Marie Luisa Schmidt3, Yu-Chi Chen1, Nadia Slepushkina1, Ari Watt2, Eric Dunham2, Kyle Shifflett2, Shelby Traeger2, Anne Leske4, Cynthia Martellaro2, Janine Brandt3, Sonja Best2, Jrgen Stech3, Stefan Finke3, Angela Rmer-Obersdrfer3, Allison Groseth2,4, Heinz Feldmann2, Thomas Hoenen2,3

1 Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD 20892, USA 2 Laboratory of Virology, Division of Intramural Research, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA

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3 Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, 17493 Greifswald Insel Riems, Germany 4 Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, 17493 Greifswald Insel Riems, Germany

Ebola viruses (EBOV) cause a severe disease in humans and non-human primates with case fatality rates of up to 90%, for which only limited experimental treatments and vaccines have been developed. The recent EBOV outbreak in West Africa highlighted the need for improved therapeutic treatment options against this virus. Approaches targeting host factors/pathways are advantageous because they may have the potential to target a wide range of viruses, including newly emerging ones, and because the development of resistance is unlikely. In order to identify such host factors, we performed a genome-wide siRNA screen against 21,597 human genes to assess their activity in EBOV genome replication and transcription. As a platform for screening and subsequent characterization of hits we used reverse-genetics based life cycle modelling systems that recapitulate these processes without the need for a maximum containment laboratory. These systems are based on minigenomes, i.e. miniature versions of the viral genome in which some or all of the viral open reading frames have been replaced with a reporter protein such as green fluorescent protein (GFP) or luciferase. In more advanced versions of these systems, called transcription-and-replication competent virus-like particle (trVLP) systems, these minigenomes are packaged into trVLPs that can infect target cells, but are biologically restricted to cells expressing multiple ebolavirus proteins in trans. Therefore, these systems provide a way to model virtually the entire EBOV life cycle (i.e. particle entry, genome replication and transcription, and progeny particle production) over multiple infectious cycles safely in a normal biosafety-level 1 environment. Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for genome replication and transcription of EBOV. Targeting this pathway with a small molecule inhibitor showed antiviral activity against EBOV both in life cycle modelling systems, as well as in experiments with infectious EBOV, and against other non-segmented negative-sense RNA viruses (NNSVs), highlighting the power of this approach.

Oral 21. Influenza A virus infection triggers IFITM3 clustering in human lung cells

Susann Kummer and Hans-Georg Krusslich

Department of Infectious Diseases, Virology, Heidelberg University

Influenza A virus (IAV) replication, as for viruses in general, depends on close interaction with host cell factors to establish infection and impede anti-viral activity. Previous studies reported a strong influence on IAV replication mediated by interferon-induced transmembrane proteins (IFITMs). Furthermore, IFITM polymorphisms were described to correlate with the severity of IAV disease in human infections. The IAV replication cycle initiates by attachment of the viral particle to the host cell plasma membrane followed by endocytic uptake and fusion. First results indicate that IFITMs act at this stage of IAV replication and are thought to block viral membrane fusion via changing cellular membrane properties. Based on these results, we aimed to study localization of IFITMs depending on IAV infection. We focused our analysis on IFITM3 in IAV infection of human epithelial lung cells (A549 cells), covering the entire infection cycle. Initial studies applying conventional confocal microscopy using overexpressed tagged proteins did not yield unequivocal results, thus we employed stimulated emission depletion (STED) super-resolution microscopy to detect endogenous IFITM3. Computational cluster analysis was used to study the change in IFITM3 signal distribution during infection progression. Using an iterative approach for cluster definition and automated data analysis, we assigned IFITM3 specific signals forming a region of dense fluorescence intensity with a size larger than 105 nm3 being one cluster. These structures initially appeared at 2-3 hours p.i. and became significant 3-5 hours p.i. in infected cells. Cluster formation is dependent on virus entry as it is blocked in IAV infection in presence of bafilomycin impeding endosomal acidification. In conclusion, we observed an unexpected re-distribution and clustering of the antiviral IFITM3 during IAV infection. Ongoing studies attempt to identify the IAV factor inducing IFITM3 clustering and to study this process in live cells at nanoscale resolution.

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Oral 22. Lifecycle modelling systems support inosine monophosphate dehydrogenase (IMPDH) as a pro-viral factor for efficient New World arenavirus RNA synthesis

Eric C. Dunham1, Anne Leske2, Kyle Shifflett1, Ari Watt1, Heinz Feldmann1, Thomas Hoenen1,3, Allison Groseth1,2

1 Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, MT, USA 2 Friedrich-Loeffler-Institut, Junior Research Group Arenavirus Biology, Greifswald - Insel Riems, Germany 3 Friedrich-Loeffler-Institut, Institute of Molecular Virology and Cell Biology, Greifswald - Insel Riems, Germany

Infection with Junn virus (JUNV) is currently being effectively managed in the endemic region using a combination of vaccination and plasma therapy. However, the long-term sustainability of that approach is unclear, and similar resources are not available for other New World arenavirus species. As a result, there has been renewed interest regarding the potential of drug-based therapies and particularly the identification of novel compounds with therapeutic potential, either for use alone or in combination with existing drugs. Here we present the establishment of JUNV life cycle modelling systems (i.e. minigenome and transcription-replication virus-like particle (trVLP) systems) with subsequent optimization to a degree suitable for high-throughput miniaturization. These systems are based on the use of viral genome analogues to model RNA synthesis (i.e. transcription and replication) by the viral ribonucleoprotein complex components safely under BSL1 conditions. Using the minigenome system, we then conducted a limited drug library screen and identified AVN-944 as an inhibitor of arenavirus transcription/replication. Like mycophenolic acid (MPA), which has also been shown to inhibit JUNV RNA synthesis, AVN-944 is thought to target de novo GTP synthesis directly as a non-competitive inosine monophosphate dehydrogenase (IMPH) inhibitor, without the additional mechanisms of action associated with other IMPDH inhibitory drugs (e.g. Ribavirin and 3f (10-allyl-6-chloro-4-methoxy-9(10H)-acridone)). Consistent with this mechanism of action, the antiviral effect of AVN-944 could be reversed with exogenous guanosine addition, while for Ribavirin this was only partly the case. In order to further validate a direct role of IMPDH, as suggested by the inhibitor data, we then used a JUNV trVLP system to evaluate the effects of IMPDH-directed siRNA knock-down. The antiviral effect of AVN-944 as well as siRNA inhibition on JUNV RNA synthesis supports that, despite playing only a minor role in the activity of ribavirin, specific non-competitive IMPDH inhibitors have significant therapeutic potential for use against arenaviruses, including New World arenaviruses. Further, this study highlights the principle suitability of arenavirus lifecycle modelling systems for the identification and mechanistic characterization of novel antiviral compounds.

Oral 23. The p24 family protein TMED10/Tmp21/p241 anchors m152/gp40 in the endoplasmic reticulum to abolish MHC class I surface expression

Venkat Raman Ramnarayan, Linda Janssen, Zeynep Hein, Natalia Lisa, Swapnil Ghanwata, and Sebastian Springer*

Department of Life Science and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany

a These authors contributed equally.

The murine cytomegalovirus immunoevasin m152/gp40 retains fully folded and assembled major histocompatibility complex class I molecules (MHC class I) in the early secretory pathway and prevents peptide presentation to CD8+ T cells. gp40 also subverts natural killer cell activation by retaining RAE-1, a ligand of the NKG2D receptor, inside the cell. We show that the linker sequence of gp40, which connects the lumenal and the transmembrane domains of this protein, is required to retain gp40 in the ER/Golgi. Using unbiased co-immunoprecipitation and mass spectrometry, we show that gp40 interacts with TMED10, a member of the p24 family of ER/Golgi transmembrane proteins. The presence of TMED10 and its localization to the early secretory pathway are required for gp40 to retain class I, since, the depletion or a mutation of the ER retention/retrieval sequence of TMED10 (namely the KKxxx

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sequence) restores normal cell surface class I levels. Our data show, for the first time, a p24 protein being exploited by a virus for immune evasion.

Oral 24. The mouse cytomegalovirus protein M25 a key player in cytomegalovirus driven cell rounding manipulates the activity of the p53 protein in infected cells

Ivana Kutle, Martina Dezeljin, Boris Bogdanow, Lder Wiebusch, Rudolf Bauerfeind, and Martin Messerle

Institute of Virology, Hannover Medical School (MHH), Hannover, Germany

Although mostly harmless for healthy individuals, cytomegalovirus (CMV) infection of immune deficient patients as well as congenitally transmitted can cause severe disease. Lytic CMV infected cells face extensive morphological changes culminating in so-called cytopathic effects (CPE). The most prominent CPE induced by mouse CMV (MCMV) is cell rounding. Infected cells undergo remodeling of the actin cytoskeleton: loos of stress fibers, formation of cortical actin and disassembly of focal adhesions. In order to clarify whether these changes occur only as host reaction to viral infection or whether they are actively triggered by the virus, a library of MCMV mutants was screened and one mutant identified that did not display the typical CPE. Disruption of the M25 ORF was found to be responsible for this phenotype. Moreover, when exogenously expressed, the M25 gene alone was able to stimulate cell rounding. Deletion of the M25 ORF also reduced infectious particle production in cell culture and diminished cell-to-cell spread, resulting in a small plaque phenotype. To understand the molecular mechanisms of M25-mediated functions we utilized co-immunoprecipitation linked to SILAC based mass spectrometry to identify host cell proteins interacting with M25. Validation of putative interaction partners implies involvement of M25 in protein de-phosphorylation, cell cycle and cell death pathways. The most interesting interaction partner of M25 in infected cells is the tumor suppressor protein p53. M25 increases the level of the 53 protein and changes its subnuclear localization. Furthermore, we found decreased transcript levels of p53 downstream target genes suggesting that M25 inhibits the transcriptional activity of p53. Our results suggest that cell rounding in MCMV infected cells is actively induced by M25 expression leading to cytoskeletal changes and increased release of infectious MCMV progeny, most likely by interference with p53-mediated regulation of the cell cycle and major cellular kinase cascade signaling.

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Poster presentations

Poster 1. Monitoring the quality and aggregation state of viral / VLP vaccine preparations using Nanoparticle Tracking Analysis (NTA)

Dr. Marco Marenchino

Malvern Instruments GmbH, Rigipsstr. 19, 71083 Herrenberg, Germany

The ability to count and size viruses and their aggregates in liquid suspension is becoming increasingly important to those involved in the development of live, attenuated, inactivated and VLP-based viral vaccines. Viruses or VLPs need to be cultured or expressed in live cells, harvested and then purified. Vaccine manufacturers are interested in monitoring the purity of the viral preparation at various key stages of the purification process and understanding the concentration of virus material present. Conventional methods like infectivity assays or protein-based assays, including ELISA are able to provide information about either infectious or total particle count, but do not reflect any state of aggregation. The aggregation state has various effects on vaccine efficacy as well as increasing evidence that larger aggregation particles lead to adverse reactions and high inter-individual aberrations. This is of importance for commercial vaccine producers as well as for academic investigators, who rely on reproducible data in small study groups in preclinical trials. Nanoparticle Tracking Analysis (NTA) is a new approach to sizing and counting viruses, which quantifies the number of viruses versus the number of virus aggregates. The technique is increasingly used to monitor aggregation state and concentration of viral vaccines. NTA is a novel light scattering method which determines nanoparticle size through simultaneously tracking and analysing the trajectories of individual particles in Brownian motion. The technique can also be integrated with fluorescence filters to allow fluorescently labelled/loaded particles to be selectively analysed. This can be of particular importancewhere the suspension is not purified. Examples how NTA has been successfully employed to quantify and monitor aggregation will be discussed with the help of case studies including influenza virus (IVA), avian flu VLP (H5N1) and Adenovirus (ADV). In addition, we will discuss an approach to measure Adeno-associated virus particles which typically fall below the LOD for individual particle tracking techniques.

Poster 2. Analysis of the interferon response in induced pluripotent stem cells

Janik Bhnke1, Denise Hbner1, Nicole Christin Bilz1, Sandra Pinkert2, Megan Stanifer3, Steeve Boulant3, Uwe Gerd Liebert1, Claudia Claus1

1 Institute of Virology, University of Leipzig, Leipzig, Germany 2 Charit - Universittsmedizin Berlin, Institut fr Biochemie, Berlin, Germany 3 University of Heidelberg, Department of Infectious Disease, Heidelberg, Germany

The effects of viral infections on human embryogenesis including interferon response mechanisms are still not completely understood. New models such as human induced pluripotent stem cells (hiPSC) are warranted for the analysis of these effects, as animal models do not include human-specific factors. hiPSCs hold the potential to cover the very early phase of human embryonic development from the blastocyst stage up to early gastrulation. This study addresses the mode of entry of recombinant coxsackievirus B3 encoding green fluorescent protein (CVB3-EGFP) and the induction of an interferon (IFN) response in hiPSCs. The expression level of the CVB3 receptors coxsackievirus and adenovirus receptor (CAR) und complement-decay accelerating factor (DAF or CD55) within hiPSC colonies was analyzed by immunofluorescence analysis. The induction of an IFN response was quantified at the protein level through the bead-based LEGENDplexTM immunoassay and at the transcript level by quantitative PCR. Pre-incubation of hiPSCs with antibodies against CAR notably reduced CVB3-EGFP infection. This indicates that CAR plays an important role in CVB3-EGFP infection of hiPSCs. While CVB3-EGFP infection itself did not evoke a notable IFN response, its infection was impaired by extracellular interferon treatment. In conclusion, interferon response mechanisms appear to be present in hiPSCs and could confer at least partial protection against viral infections.

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Poster 3. Drug resistance and phylogenetic analysis of protease gene of treatment nave HIV-1 infected injecting drug users in Pakistan

Saima Yaqub1, Tahir Yaqub1, Nadia Mukhtar2, Zarfishan Tahir3, Ali Jan Khan4, Muhammad Zubair Shabbir5, Asif Nadeem6, Firnas Ata Ur Rehman1, Shahid Nawaz1, Muzaffar Ali1, Muhammad Furqan Shahid1

1 Department of Microbiology, University of Veterinary and Animal Sciences Lahore, Pakistan 2 Punjab AIDS Control Program, Primary & Secondary Health Care Department, Government of Punjab, Pakistan 3 Director General Health Services, Primary and Secondary Health Department, Government of Punjab, Pakistan 4 Primary and Secondary Healthcare Department Government of the Punjab, Pakistan 5 Quality Operations Laboratory, University of Veterinary and Animal Sciences Lahore, Pakistan 6 Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore, Pakistan

BACKGROUND: Many different subtypes of HIV-1 have been reported in Pakistan by analyzing viral gene sequences. Little information is available about the dissemination of circulating recombinant forms of HIV type 1 prevailing among injecting drug users (IDUs) in Lahore, Pakistan.

OBJECTIVES: To analyze the drug resistance and circulating subtype distribution among treatment naive HIV-1 infected IDUs in Lahore, Pakistan.

METHODS: Plasma sample were obtained from 11 HIV-1 infected newly diagnosed IDUs and RNA was extracted. Reverse transcriptase based PCR was performed and PCR products were sequenced. Later on, HIV-1 subtype and drug resistance mutations were analyzed at Protease region of HIV-1 using phylogenetic analysis and Stanford HIV drug resistance database respectively.

RESULTS: Phylogenetic analysis of newly characterized sequences of HIV-1 protease gene showed that CRF02_AG is present. The analysis of drug resistance mutations (DRM) showed no major DRM to protease inhibitors among studied subjects.

CONCLUSION: Our study confirms the predominance of CRF02_AG among HIV-1 infected IDUs in Lahore, Pakistan. As the HIV-1 prevalence continues to rise in Pakistan among different risk communities, there is need to perform well designed surveillance studies for continuously monitoring circulating subtypes as well as level of drug resistance for effective therapeutic approaches.

Poster 4. mCMV immunoevasin gp40 downregulates cellular stress marker RAE-1

Natalia Lis, Swapnil Ghanwat, Venkat Raman Ramnarayan, Sebastian Springer

Department of Life Sciences and Chemistry, Jacobs University Bremen, Germany

Retinoic acid early-inducible protein 1- gamma (RAE-1) is a ligand for NKG2D, a natural killer (NK)

cell activating receptor. The cell surface levels of RAE-1 is one of the most important factors that determine the activation of NK cells during herpesvirus infection. The murine cytomegalovirus (mCMV) glycoprotein gp40 (encoded by the m152 gene) downmodulates the cell surface

expression of RAE-1, and thus limits the innate immune response against the virus. gp40

presumably binds to RAE-1 and retains it in the early secretory pathway. It remains unclear how

gp40 prevents the export of RAE1-, since gp40 lacks any known ER-retention sequence. We

propose that the viral protein either prevents proper maturation of RAE-1 upon binding, or tethers

RAE-1 to another cellular retention protein to arrest it in the early secretory pathway. Investigating the interaction and retention mechanisms will enable us to better understand the processes of protein maturation and trafficking, it might also help us identify a novel cellular retention factor.

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Poster 5. Immunization with immune complexes modulates the fine-specificity of antibody responses to a Flavivirus antigen

Georgios Tsouchnikas1, Jrgen Zlatkovic1, Johanna Jarmer1, Judith Strauss1, Oksana Vratskikh1, Michael Kundi2, Karin Stiasny1, Franz X. Heinz1

1 Department of Virology, Medical University of Vienna, Vienna, Austria 2 Institute of Environmental Health, Medical University of Vienna, Vienna, Austria

Immune complexes (ICs) can modulate the immune response to the antigen via several antibody feedback-mechanisms, resulting in enhancement or suppression. However, there is less information about the capacity of ICs to alter the epitope-specificity of the antibody response. Since in polyclonal sera, the specificity of antibodies to different antigenic sites can affect functional activity, variations can be relevant for an effective immune response. The objective of this mouse immunization-study was to investigate changes in quantity and fine-specificity of the antibody response to the tick-borne encephalitis (TBE) virus envelope (E) protein administered alone or as an IC with monoclonal antibodies (mAbs) directed to its structural domains (DI, DII, DIII). Due to the modular organization of E, the fine-specificity of polyclonal antibodies can be dissected by immunoassays using recombinant single domains or domain-combinations of E. Our analyses revealed that immunization with the IC containing a monoclonal antibody specific for DII strongly modulated the fine-specificity of the antibody. These results could be explained mechanistically by demonstrating that this mAb dissociates the E dimer into monomers, and thus exposes new immunogenic epitopes. A different mechanism was found for a DIII-specific mAb which reduced the response to the virion by shielding its highly surface-accessible epitope. Our results provide direct mechanistic insights into the structure-specific modulation of an immunogen by bound antibodies that lead to a change in the specificity of polyclonal antibody responses. Similar phenomena can play also a role in a natural situation in which pre-existing antibodies encounter the antigen and form ICs in vivo.

Poster 6. Entry of Merkel cell polyomavirus into A549 Cells

Miriam Becker1,3, Melissa Dominguez1,3, Lilo Greune2,3, Nicole Cordes1,3, Esther Weghake1,3, Rachel Schowalter4, Christopher B. Buck4, M. Alexander Schmidt2,3, and Mario Schelhaas1,3

1 Section for Cellular Virology, Institutes of Virology and Medical Biochemistry, ZMBE, University of Munster, Germany 2 Institute of Infectiology, ZMBE, University of Munster, Germany 3 Cluster of Excellence EXC1003, Cells in Motion, CiM, Munster, Germany 4 Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

Merkel Cell Polyomavirus (MCPyV) is a member of the polyomaviridae and was recently discovered in an aggressive form of skin cancer known as Merkel cell carcinoma (MCC). Although reports indicate that MCPyV virions are shed from human skin, the precise cellular tropism of the virus in healthy subjects remains unclear, in particular since MCPyV DNA has also been detected in samples from a variety of other tissues. MCPyV is also different from other polyomaviruses in that initial attachment requires sulfated polysaccharides such as heparan sulfates and/or chondroitin sulfates, before the virus engages sialic acid residues as a co-receptor like other polyomaviruses. To explore the question, whether MCPyV differs significantly in its entry process from other polyomaviruses, we analyzed the cell biological determinants of MCPyV vectors (pseudoviruses) into A549 cells, a highly transducable lung carcinoma cell line, in comparison to Simian Virus 40, a well-studied polyomavirus, human papillomavirus type 16, and other control viruses. Our results indicated that MCPyV enters cells via caveolar/raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or glycosphingolipid-enriched carriers. The viruses internalized in small, tight fitting endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Trafficking required microtubular transport, acidification of endosomes, and a functional redox environment such as other polyomaviruses. Based on the fact that only minor amounts of viruses reached the ER and that the majority of viruses appeared to be stuck in endosomal compartments, we suggest the trafficking from endosomes to the ER is a bottleneck of initial infection of A549 cells.

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Poster 7. Early phlebovirus host cell interactions using Uukuniemi virus as a model

Anja Hoffmann, Hannah Fleckenstein, Malte Simon, Sven Blobner, Jana Koch, Claudia Robens, Dr. Pierre-Yves Lozach

Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany

Phleboviruses are transmitted by arthropods and several members cause severe diseases in livestock and humans, as for example Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV) and Rift Valley Fever Virus (RVFV). Thus far endocytic pathways and host factors used by these emerging pathogens to infect cells remain largely uncharacterized and currently no specific treatment is approved for human use. Preventing phlebovirus infection requires approaches aiming to target early virus-host cell interactions prior to genome release. Phleboviruses are late-penetrating viruses, a large group of viruses that rely on late endosomal compartments for productive penetration. To reach those late endosomal compartments Uukuniemi virus (UUKV), a prototype phlebovirus, relies on Rab5+ early endosomes while Rab7, a small GTPase critical for maturation of late endosomes, does not seem to be required. A genome wide siRNA screen performed in our lab identified VAMP3 as a novel host cellular factor required for late penetration of UUKV. Among other functions, VAMP3 was proposed to mediate initiation of autophagy and fusion between multivesicular bodies and autophagosomes, thereby bridging the autophagosomal and endocytic pathway. Interestingly autophagy related genes WIPI1, Rab1b, Rab11a and FIP200 were also found in the list of potential host factor candidates from the siRNA screen leading to the assumption that autophagy plays a role in UUKV infection. Our results suggest that the key autophagy protein Atg7 as well as the autophagy-related small GTPases Rab1b and Rab11a are involved in UUKV infection. SiRNA-mediated silencing of all three proteins as well as expression of dominant negative mutants of Rab1b and Rab11a inhibit UUKV infection. Currently we focus on the development of a fusion assay with the goal to clarify in which step of infection autophagy-related proteins play a role.

Poster 8. Establishment of a single-point assay to evaluate the relationship of molecular structure and antiviral activity of cationic amphiphilic drugs

Antonia P. Gunesch1,2, Francisco J. Zapatero-Belinchon1,2, Michael P. Manns2, Eike Steinmann3, Mark Brnstrup4, Thomas von Hahn1,2

1 Institute for Molecular Biology, Hannover Medical School, Hannover, Germany 2 Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany 3 Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hannover, Germany 4 Department of Chemical Biology, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany

Enveloped virus families Filovidirae, Arenaviridae, Rhabdoviridae, Coronaviridae, Togaviridae, Flaviviridae and Bunyaviridae enter host cells via endosomal trafficking and an acidic pH-dependent early or late fusion event between viral envelope and host endosomal membrane. The lack of effective antiviral treatment or vaccination makes novel antiviral drug development necessary in order to alleviate morbidity and mortality in epidemics. Cationic amphiphilic drugs (CADs) were found to have broad antiviral properties but there are also concerns about adverse effects in host cells, such as drug-induced phospholipidosis. Nevertheless, the antiviral mechanism of action and structure-function relationship of CADs is yet unclear. In this project, we aim to identify candidates with high, intermediate, and low antiviral activity levels for structure-function analysis with the goal to identify structural determinants of CAD antiviral activity. Concentration-dependent inhibition of viral entry by the prototypical antiviral CAD amiodarone was determined in human endothelium/lung hybrid (EAhy 926) cells with lentiviral particles harboring envelope glycoproteins of Lassa virus, Guanarito virus, Marburg virus, vesicular stomatitis virus or chikungunya virus as well as a luciferase reporter gene. Upon determination of the best-suited pseudovirion for our drug screening system, we determined the specific IC 50 of amiodarone. These parameters were then tested on EAhy 926, HEP-3B and HeLa cells for selection of a host cell line. Simultaneously, cytotoxicity was validated. With these defined conditions a single-point

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assay was established facilitating screening of a panel of CADs with various physicochemical properties. Standardization of molar concentration to pply to all CADs in the screen was performed by cytotoxicity and viral inhibition assays using amiodarone. For amiodarone, IC 50 = 5 M and CC 50 = 28.7 M were determined. Thus, we chose 5 M as an optimal single-point CAD concentration. The cell line EAhy 926 was selected for subsequent assays due to its wide susceptibility to all tested pseudoviruses. In specific, MARV showed a high transduction frequency and clear inhibition by 5 M amiodarone, and was picked as representative amiodarone-sensitive pseudovirus. In an initial single-point screening experiment we evaluated 14 out of a panel of 47 CADs, of which four showed antiviral activity similar to amiodarone. Furthermore, these four candidates did not show a cytotoxic effect on host cells. In conclusion, a single-point drug screening assay based on infection of EAhy 926 host cells with MARV was established to evaluate antiviral activity of CADs. Initial experiments indicate that there are differences among CADs with regard to their antiviral potency. Subsequently, drug-induced phospholipidosis has to be monitored aiming to evaluate host cell effects of CADs. Continuing from this point, initial structure-function dependencies will be presented.

Poster 9. The chaperone dynein LL1 mediates cytoplasmic transport of empty and mature hepatitis B virus capsids

Lara Gallucci1, Quentin Osseman1, Marie-Lise Blondot1 and Michael Kann1

1 MFP CNRS UMR 5234, University of Bordeaux, France

Hepatitis B virus (HBV) has a DNA genome but it replicates within nucleus via reverse transcription. Genome maturation from RNA to DNA occurs in cytoplasmic capsids, which are formed from the assembly of 240 copies of the capsid protein core (Cp) around the viral RNA pregenome and the viral polymerase. Mature capsids then become enveloped by the viral surface proteins or they are transported to the nucleus leading to nuclear genome accumulation. Cp are overexpressed relative to their abundance in virions and the super numerous Cp assemble to empty cytosolic capsids. Capsids in this compartment are correlated with inflammation and epitopes of the capsid protein core (Cp) are the main target for T cell-mediated immune response and removal of capsids from the cytosol is consequently assumed to be beneficial for HBV infection. We investigated the mechanism of retrograde cytoplasmic capsid transport, using cytosolic microinjection of mature, empty and RNA-containing capsids. Pull-down assays revealed that only transport-competent capsids interact with the dynein light chain LL1 (DynLL1). In the dynein motor complex this chain stabilizes the dynein intermediate chains (DynIC) and we could reconstitute a complex of capsids with DynLL1 and DynIC. In vitro binding assays to microtubules and co microinjections of capsids with an excess of DynLL1 and with mutant DynLL1, which was found to be unable to bind to DynIC, confirmed that capsid attachment to DynLL1 is the dominant if not the only interaction leading to retrograde capsid transport.

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List of participants and email contact addresses

Allison Groseth allison.groseth@fli.de

Ana Hickford Martinez hickfordmartinez.ana@mh-hannover.de

Ana Nascimento Nascimento.Ana@mh-hannover.de

Anja Hoffmann anja.hoffmann@uni-heidelberg.de

Anja Schbel anja.schoebel@leibniz-hpi.de

Annica Flemming annica.flemming@med.uni-heidelberg.de

Antonia-Patricia Gunesch gunesch.antonia-patricia@mh-hannover.de

Ari Helenius ari.helenius@bc.biol.ethz.ch

Beate Sodeik Sodeik.Beate@mh-hannover.de

Christin Mller christin.mueller@viro.med.uni-giessen.de

Claudia Claus claudia.claus@medizin.uni-leipzig.de

Daniela Freytag Daniela.Freytag@bd.com

Eva Herker eva.herker@leibniz-hpi.de

Floriane Lagadec floriane.lagadec@u-bordeaux.fr

Francisco Zapatero-Belinchon ZapateroBelinchon.Francisco@mh-hannover.de

Georgios Tsouchnikas virologie@meduniwien.ac.at

Ghanwat Swapnil s.ghanwat@jacobs-university.de

Gisa Gerold gisa.gerold@twincore.de

Hanna Bley hanna.bley@leibniz-hpi.de

Holda Anagho anagho@stud.uni-heidelberg.de

Iris Medits virologie@meduniwien.ac.at

Ivana Kutle Kutle.Ivana@mh-hannover.de

Janik Bhnke janik.boehnke@medizin.uni-leipzig.de

Joao Miguel Freire joao-miguel.freire@pasteur.fr

Jrg Nesch jpf.nuesch@dkfz.de

Lara Gallucci lara.gallucci@u-bordeaux.fr

Laura Soria-Martinez laurasoria@uni-muenster.de

mailto:Nascimento.Ana@mh-hannover.demailto:anja.hoffmann@uni-heidelberg.demailto:anja.schoebel@leibniz-hpi.demailto:annica.flemming@med.uni-heidelberg.demailto:gunesch.antonia-patricia@mh-hannover.demailto:ari.helenius@bc.biol.ethz.chmailto:Sodeik.Beate@mh-hannover.demailto:christin.mueller@viro.med.uni-giessen.demailto:claudia.claus@medizin.uni-leipzig.demailto:Daniela.Freytag@bd.commailto:eva.herker@leibniz-hpi.demailto:floriane.lagadec@u-bordeaux.frmailto:ZapateroBelinchon.Francisco@mh-hannover.demailto:virologie@meduniwien.ac.atmailto:s.ghanwat@jacobs-university.demailto:gisa.gerold@twincore.demailto:hanna.bley@leibniz-hpi.demailto:anagho@stud.uni-heidelberg.demailto:virologie@meduniwien.ac.atmailto:Kutle.Ivana@mh-hannover.demailto:janik.boehnke@medizin.uni-leipzig.demailto:joao-miguel.freire@pasteur.frmailto:jpf.nuesch@dkfz.demailto:lara.gallucci@u-bordeaux.frmailto:laurasoria@uni-muenster.de

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Lisa Lasswitz lisa.lasswitz@twincore.de

Luis Jaime Vallejo Castano Vallejo@stud.uni-heidelberg.de

Magalie Mazelier magalie.mazelier@med.uni-heidelberg.de

Marco Marenchino marco.marenchino@malvern.com

Margaret Kielian margaret.kielian@einstein.yu.edu

Mario Schelhaas schelhaas@uni-muenster.de

Marta Fratini m.fratini@dkfz-heidelberg.de

Melina Vallbracht Melina.Vallbracht@fli.de

Miriam Becker miriam.becker@uni-muenster.de

Natalia Lis n.lis@jacobs-university.de

Nicole Christin Bilz christin.emmrich@medizin.uni-leipzig.de

Olaf Isken isken@vuz.uni-luebeck.de

Pia Brinkert pia.brinkert@uni-muenster.de

Pierre-Yves Lozach pierre-yves.lozach@med.uni-heidelberg.de

Saima Yaqub 2014-phd-1013@uvas.edu.pk

Sarai Rodrguez Navarro sarai.rodriguez@mdpi.com

Sebastian Joecks Sebastian.Joecks@twincore.de

Serrero Manutea manuteaser@hotmail.com

Steeve Boulant s.boulant@dkfz.de

Stefan Phlmann SPoehlmann@dpz.eu

Susann Kummer susann.kummer@med.uni-heidelberg.de

Thomas Hoenen thomas.hoenen@fli.de

Venkat Raman Ramnarayan v.ramnarayan@jacobs-university.de

mailto:lisa.lasswitz@twincore.demailto:Vallejo@stud.uni-heidelberg.demailto:magalie.mazelier@med.uni-heidelberg.demailto:marco.marenchino@malvern.commailto:margaret.kielian@einstein.yu.edumailto:schelhaas@uni-muenster.demailto:m.fratini@dkfz-heidelberg.demailto:Melina.Vallbracht@fli.demailto:miriam.becker@uni-muenster.demailto:n.lis@jacobs-university.demailto:christin.emmrich@medizin.uni-leipzig.demailto:isken@vuz.uni-luebeck.demailto:pia.brinkert@uni-muenster.demailto:pierre-yves.lozach@med.uni-heidelberg.demailto:2014-phd-1013@uvas.edu.pkmailto:Sebastian.Joecks@twincore.demailto:manuteaser@hotmail.commailto:s.boulant@dkfz.demailto:SPoehlmann@dpz.eumailto:susann.kummer@med.un