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18th Workshop
"Cell Biology of Viral Infections"
of the German Research Platform for Zoonoses &
the Society for Virology (GfV)
"Cell biology of zoonotic viral infections: from reservoirs to humans"
Kloster Schöntal
Oct. 23 – 25, 2019
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Cover shows syncitia resulting from the cell-cell fusion mediated by Uukuniemi virus (“fusion-from-without”). Nuclei appear in blue and cytoplasm in red. Photo courtesy of Lozach lab.
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We thank the following organizations and companies for the financial support of our workshop:
German Research Platform for Zoonoses Berlin, Münster, and Greifswald, Germany http://www.zoonosen.net/
Chica and Heinz Schaller (CHS) Foundation Heidelberg, Germany http://www.chs-stiftung.de
Gesellschaft für Virologie e.V. Ulm, Germany http://www.g-f-v.org
Reblikon GmbH Schriescheim, Germany
Deutsche Gesellschaft für Zellbiologie Heidelberg, Germany http://zellbiologie.de/
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Workshop Program
Day 1 (Wednesday, October 23, 2019)
10:30 Bus Departure from Würzburg
12:00 – 13:25 Welcome Lunch
13:25 – 13:30 Opening and Welcome Remarks Gisa Gerold and Pierre-Yves Lozach
13:30 – 14:30 Keynote Lecture 1 “Exploring the diversity of mosquito-borne viruses” PD Sandra Junglen Charité Universitätsmedezin, Berlin, Germany
14:30 – 17:00 Workshop Session 1 – Virus Entry and Intracellular Trafficking
Oral 1. Jared Kirui, Twincore, Hannover
“The phosphatidylserine receptor TIM-1 is an entry factor for Chikungunya Virus”
Oral 2. Hannah Kleine-Weber, University Göttingen
“Naturally occurring polymorphisms in dipeptidyl-peptidase 4 can reduce MERS-coronavirus entry into host cells”
Oral 3. Jana Koch, University Hospital Heidelberg
“Entry of Toscana virus into Mammalian Host Cells”
15:30 – 16:00 Coffee Break
Oral 4. Sophie Winter, Heidelberg University
“Cryo-electron tomography reveals pH-dependent structural rearrangements of Ebola VLPs”
Oral 5. Steffen Klein, Heidelberg University
“Influenza A virus entry and IFITM3 induced inhibition of membrane fusion studied by cryo-CLEM”
Oral 6. Melina Vallbracht, Friedrich-Löffler-Institut, Riems
“So close and yet so different: Comparing the complex fusion machinery of the alphaherpesviruses Pseudorabies virus and Herpes simplex virus”
17:00 – 17:15 Short Break
17:15 – 18:15 Special Lecture – Scientific editing “Combatting “fake news”: how to structure and disseminate research
findings to effectively reach your targeted audience” Prof. Karen Mossman
MacMaster University, Hamilton, Canada
18:30 Social Event, dinner
19:30 Wine Tasting in the Cellar
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Day 2 (Thursday, October 24, 2019)
7:30 – 9:00 Breakfast
09:00 – 10:00 Keynote Lecture 2 “Reaching borders: intracellular transport of filoviruses”
Prof. Stephan Becker University of Marburg, Germany
10:00 – 12:30 Workshop Session 2 – Virus Replication, Assembly, and Egress
Oral 7. Lucie Fénéant, Friedrich-Löffler-Institut, Riems
“Generation of reporter expressing New-World Arenaviruses: a systematic comparison”
Oral 8. Yasutsugu Suzuki, Pasteur Institute, Paris, France
“Functional investigation of endogenous flaviviral elements in Aedes mosquitoes”
10:40 – 11:10 Coffee Break
Oral 9. Anja Schöbel, University of Marburg
“Molecular interactions of HCV core and the long interspersed nuclear element 1 (LINE1) ORF1 protein at HCV assembly sites”
Oral 10. Nico Becker, University of Marburg
“Nipah virus matrix protein critically determines inclusion body formation”
Oral 11. Jacomine Krijnse-Locker, Pasteur Institute, Paris, France
“Membrane assembly of large DNA viruses; electron microscopy leads the way”
Oral 12. Hannah Bley, University of Marburg
“Identification of Annexin A3- and Perilipin 2-interacting proteins in hepatitis C virus-infected cells using the proximity labeling methods bioID2 and APEX2”
12:30 – 13:30 Lunch
13:30 – 13:45 Group Picture
13:45 – 15:00 “Kloster Schöntal” Tour
15:00 – 16:20 Workshop Session 3 – Host Cell – Virus Interactions
Oral 13. Francisco Zapatero, Twincore, Hannover
“The effects of lipoprotein receptors and lipid-lowering drugs on the HCV replication cycle”
Oral 14. Linus Bostedt, Friedrich-Löffler-Institut, Riems
“Unravelling the activities of Junín virus nucleoprotein isoforms in the viral life cycle”
Oral 15. Zina Uckeley, University Hospital Heidelberg
“Quantitative proteomics of Uukuniemi virus-host cell interactions reveals GBF1 as proviral host factor for phleboviruses”
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Oral 16. Lisa Wendt, Friedrich-Löffler-Institut, Riems
“The Ebola virus nucleoprotein NP recruits the nuclear RNA export factor NXF1 for viral mRNA export from inclusion bodies”
16:20 – 18:15 Poster Session (coffee will be served)
18:15 – 19:15 Keynote Lecture 3 “Tick cell lines: tools for zoonotic virus research“ Dr Lesley Bell-Sakyi University of Liverpool, United Kingdom
19:30 Workshop Gala Dinner
Day 3 (Friday, October 25, 2019)
7:30 – 9:00 Breakfast
09:00 – 10:00 Keynote Lecture 4 “Zika virus interactions with innate immunity and neurotropism” Prof. Alain Kohl
MRC-University of Glasgow, United Kingdom
10:00 – 12:20 Workshop Session 4 – Antiviral and Stress Response
Oral 17. Sabrina Johanna Fletcher, Pasteur Institute, Paris, France
“Finding the elusive dsRNA receptor involved in antiviral immune response in insects”
Oral 18. Prerna Arora, University of Göttingen
“Analysis of determinants controlling antiviral activity of defective interfering influenza A virus RNAs”
10:40 – 11:10 Coffee Break
Oral 19. Johanna Wildemann, Paul-Ehrlich-Institut, Langen
“Innate immune response to Ebola virus”
Oral 20. Veronika Breitkopf, University of Veterinary Medicine, Hannover
“Molecular interactions of tick-borne encephalitis virus proteins and their role in the ER-stress response”
Oral 21. Julia Holzerland, Friedrich-Loeffler-Institut, Riems
“Bad, Noxa and Puma are key regulators of Tacaribe virus-induced apoptosis”
12:10 – 12:20 Prize of the Best Oral and Poster presentations, Closing Remarks Gisa Gerold and Pierre-Yves Lozach
12:30 – 14:00 Lunch
14:00 Bus departure to Würzburg
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Oral presentations
Oral 1. The phosphatidylserine receptor TIM-1 is an entry factor for Chikungunya Virus
Jared Kirui1, Lisa Lasswitz1, Francisco Zapatero1, Sergej Franz1, Christine Goffinet3, Graham Simmons4,5, Thomas Pietschmann1, Gisa Gerold1,2
1 Insitute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany 2 Department of Clinical Microbiology, Virology & Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, Umea, Sweden 3 Institute of Virology-Charité - Universitätsmedizin Berlin, Germany 4 Blood Systems Research Institute, San Francisco, California 5 Department of Laboratory Medicine, University of California, San Francisco
Chikungunya virus (CHIKV) is transmitted to humans by mosquitoes and can cause chronic and incapacitating arthralgia. In the past five decades numerous outbreaks have affected millions of people yet there are no approved vaccines or specific antivirals. To date, the CHIKV cell entry process is insufficiently understood. Mxra8, a recently described attachment factor for CHIKV is absent on some susceptible cells, suggesting that additional entry factors exist. Lentiviral pseudoparticle experiments imply a role for the phosphatidylserine receptors T-cell immunoglobulin and mucin domain 1 (TIM-1) in CHIKV entry. However, the relevance of TIM-1 in authentic CHIKV cell entry remained enigmatic and therefore, we aimed to characterize its role in CHIKV infection. We initially determined expression levels of TIM-1 and Mxra8 by surface antibody staining and flow cytometry in human dermal fibroblasts, kidney epithelial (HEK293T), osteosarcoma (U2-OS) and hepatoma cells (Huh-7.5). To delineate the role of phosphatidylserine receptors, we generated HEK 293T cells overexpressing wild type TIM-1 or TIM-1 with mutations in the ectodomain and cytoplasmic domain. We then infected the cells with either authentic West African or East Central South African lineage strains of CHIKV using different multiplicity of infection (MOI) and performed entry kinetic experiments. We show that authentic CHIKV efficiently infects cells lacking Mxra8, confirming the possibility of additional entry factors. In an overexpression setting, TIM-1 enhanced authentic CHIKV infection at low (0.001) to intermediate (0.1) MOI. Furthermore, we observed equal CHIKV entry kinetics between TIM-1 over expressing cells and control parental cells. This result suggests that TIM-1 only enhances CHIKV entry without altering the entry kinetics. The susceptibility of cells overexpressing TIM-1 with a double mutation (N114A and D115A) in the immunoglobulin (Ig) V domain equaled that of the parental cells. In contrast, the double mutation (K338R and K346R) in the cytoplasmic motif associated with ubiquitination or the deletion of the cytoplasmic domain hardly changed the entry enhancement of CHIKV by TIM-1 expression. Taken together, these results suggest that TIM-1 is a host factor for CHIKV cell entry and that the metal ion-dependent ligand binding site, MILIBS is important for TIM-1 function in the context of CHIKV infection. Eventually, our work will reveal molecular functions of TIM-1 in CHIKV infection and may lead to the development of specific antivirals for treatment of Chikungunya fever.
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Oral 2. Naturally occurring polymorphisms in dipeptidyl-peptidase 4 can reduce MERS-coronavirus entry into host cells
Hannah Kleine-Weber1,2, Simon Schroeder3, Nadine Krüger4,5, Christian Drosten3,6, Marcel A. Müller3,6, Stefan Pöhlmann1,2, Markus Hoffmann1
1 Infection Biology Unit, German Primate Center, Göttingen, Germany 2 Faculty of Biology and Psychology, University Göttingen, Göttingen, Germany 3 Charité-Universitätsmedizin Berlin, corporate member of FreieUniversität Berlin, Humboldt-Universitätzu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin, Germany 4 Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany 5 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany 6 German Centre for Infection Research, associated partner Charité, Berlin, Germany
Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in the Middle East but has pandemic potential. The host cell entry of MERS-CoV is facilitated bythe viral spike glycoprotein (S), which interacts with the host cell receptor dipeptidyl-peptidase 4(DPP4, CD26). DPP4 harbors a receptor binding domain which interacts with known residues in MERS-S. We analyzed public databases for polymorphisms that alter DPP4 residues interacting with MERS-S and we determined the impact of these polymorphisms on S protein-driven entry. We found that nine out of fifteen DPP4 residues that interact with MERS-S are polymorphic and binding studies revealed that polymorphisms K267E, K267N and A291P reduce MERS-S binding to DPP4 and MERS-S-driven entry without interfering with DPP4 expression. Furthermore, K267E and A291P reduced replication of MERS-CoV in DPP4-transfected cells. Collectively, our data show that naturally occurring DPP4 polymorphisms can reduce MERS-S-driven host cell entry and viral replication.
Keywords: MERS-CoV, Spike glycoprotein, DPP4, Polymorphism
Oral 3. Entry of Toscana virus into mammalian host cells
Jana Koch1, Martin Obr2, Susann Kummer1, Anja Hoffmann1 and Pierre-Yves Lozach1,3
1 CellNetworks – Cluster of Excellence and Center for Integrative Infectious Disease Research, Virology, University Hospital Heidelberg, Germany 2 Institute of Science and Technology Austria, Klosterneuburg, Austria 3 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, 50 Av. Tony Garnier, 69007 Lyon, France
Bunyavirales is a large order of RNA viruses with over 400 members worldwide. Many cause severe diseases in animals and humans. In this order, Toscana virus (TOSV) is a sandfly-borne phlebovirus present in the Mediterranean basin where it causes febrile illnesses and central nervous system infections in human. Being one of the most prevalent causes of human meningitis and encephalitis in Southern Europe during the summer season, TOSV is currently regarded as the most public-health relevant sandfly-borne phlebovirus. Despite its neuro-invasiveness and emergence, TOSV is still a neglected pathogen and little is known about its infection at the molecular and cellular levels. Using sensitive, quantitative assays based on flow cytometry, fluorimetry as well as a combination of microscopy-based techniques, we investigated early steps of TOSV life cycle and its early virus-host cell interactions. After entry into A549 cells, TOSV travelled through early endosomes in a Rab5-dependent manner and is delivered to Rab7+ and LAMP1+ late endosomes. For penetration, the acid-activated viral membrane fusion occurred within 50 min with a half-maximal time of 15 min post-entry and with a pH threshold for fusion of pH 5.5. The acid-activated penetration of TOSV required maturation from early to late endosomes. In conclusion, our data suggest that TOSV trafficked along the endosomal route and penetrated host cells from late endosomes by acid-activated membrane fusion.
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Oral 4. Cryo-electron tomography reveals pH-dependent structural rearrangements of Ebola VLPs
Sophie Winter, Androniki Kolovou, Petr Chlanda
Ebola virus (EBOV) is an enveloped negative-stranded RNA virus that has caused severe disease outbreaks in Western and Central African countries. EBOV particles are filamentous in shape and up to several microns long. The filamentous morphology is determined by the viral major matrix protein VP40 that forms a helical scaffold underneath the viral membrane and encapsulates the viral genome containing nucleocapsid. A prerequisite for virus replication is the entry into host cells to access the host cell machinery required for the formation of progeny virions. The Ebola virions are macropinocytosed and trafficked to the late endosome, where low pH activated host cell proteases cleave off the glycosylated mucin-like domain of the Ebola glycoprotein (GP), thereby exposing a fusion peptide and receptor-binding domain. The cleaved GP then binds to the endosomal cholesterol transporter NPC1 and membrane fusion takes place. Although a general entry mechanism has been proposed for EBOV, it is currently unknown how the long filamentous virions disassemble and deliver their genome into the cytoplasm. We aim to structurally characterize Ebola-virus like particles (VLPs) in vitro by cryo-electron tomography (cryo-ET) and analyze potential structural differences between particles at neutral pH and at low pH mimicking the endosome luminal environment. Subtomogram averaging (STA) of the VP40 helical layer underneath the membrane at neutral pH and at low pH will be employed to further characterize structural changes. Ebola VLPs were produced by overexpressing Ebola structural proteins in 293T cells and subsequent purification of particles from the surrounding medium by differential centrifugation. The VLPs were incubated at neutral and low pH for 30 min at 37°C prior to plunge-freezing and acquiring cryo-electron tomograms to analyze overall structural changes. Ebola VLPs resolved by cryo-ET at neutral pH have a characteristic filamentous morphology with an average diameter of 90 nm. The observed structural features of the VLPs are in agreement with those previously published in cryo-ET studies on Ebola virions. STA of the VP40 layer at neutral pH revealed the helical arrangement of VP40 dimers lining the inner leaflet of the membrane. In contrast, VLPs incubated at low pH were more variable in diameter and some of the particles lost the filamentous morphology. The majority of VLPs subjected to low pH had a detached VP40 layer and contained luminal aggregates, presumably composed of disassembled VP40. In vitro, VLPs at low pH partially loose the Ebola-characteristic filamentous morphology as a result of VP40 layer detachment from the VLP envelope. Time-lapse confocal microscopy of VLPs containing VP40-phluorin shows that VLP lumen is acidified after the pH of the surrounding buffer is lowered. We propose that VP40 layer disassembly is a critical intermediate step of viral entry that occurs prior to or concomitant with membrane fusion. How the particle’s lumen gets acidified remains to be elucidated and could reveal a yet unknown factor important in Ebola virus disassembly.
Oral 5. Influenza A virus entry and IFITM3 induced inhibition of membrane fusion studied by cryo-CLEM
Steffen Klein, Androniki Kolovou, Benedikt Wimmer, Petr Chlanda
Influenza A virus (IAV) is an important human pathogen assembling into pleomorphic, enveloped virions that enter the host cell by endocytosis. The low pH in late endosomes triggers structural changes in the viral glycoprotein hemagglutinin, facilitating viral and endosomal membrane fusion that leads to the release of the ribonucleoproteins. Viral membrane fusion is inhibited by the host protein Interferon-induced transmembrane protein 3 (IFITM3) as part of the cell-induced anti-viral response. It has been proposed that IFITM3 is able to block IAV membrane fusion in late endosomes at the stage of hemifusion, however, IFITM3 inhibition of membrane fusion has neither been studied at the molecular level nor has it been structurally examined in the context of the cell. The aim of this project is to apply a correlative light and electron microscopy (CLEM) workflow to structurally characterize IAV entry, as well as IFITM3 induced inhibition of IAV membrane fusion in human cells. To localize viral entry events in the host cell, virus-like particles (VLP) were fluorescently labelled by M1-pHluorin (pH-sensitive GFP variant) that is incorporated into the VLP during particle assembly. Purified VLPs were mixed with fluorescent beads and plunge frozen on carbon-coated grids and the frozen grids were mapped by cryoLM. The signal of the beads was used for correlation with the cryoEM map to localize virions for tomogram acquisition. CryoET
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revealed that VLPs containing M1-pHluorin have a similar morphology as wild-type VLPs, thus can be used for CLEM workflow. Human A549 lung cells expressing IFITM3-neonGreen were labelled with a lipid droplet marker and either grown on gold carbon-coated grids or sapphire disks for cryo-CLEM or CLEM using high pressure freezing and freeze substitution, respectively. CLEM revealed that the intensity of IFITM3-neoGreen fluorescent signal corresponds to multivesicular bodies (MVBs) with a high number of intraluminal vesicles. In addition, cryoET of MVBs inside IFITM3-neonGreen expressing cells revealed crystalline lipidic structures resembling previously reported cholesterol-enriched crystals.
Oral 6. So close and yet so different: comparing the complex fusion machinery of the alphaherpesviruses pseudorabies virus and Herpes simplex virus
Melina Vallbracht1, Barbara G. Klupp1, Felix Rey2, Marija Backovic2, Thomas C. Mettenleiter1
1 Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany 2 Pasteur Institute, Paris, France
Enveloped viruses rely on membrane fusion for entry and direct cell-to-cell spread. While many viruses require only one protein to mediate attachment and membrane fusion, herpesviruses utilize a more complex machinery. The prototypic human alphaherpesviruses Herpes simplex viruses 1 and 2 (HSV-1 and -2) and the porcine Pseudorabies virus (PrV) require at least four different envelope glycoproteins (g): The bona fide fusion protein gB and the heterodimeric gH/gL regulator, constituting the “core fusion machinery” conserved in all herpesviruses, and the subfamily specific receptor binding protein gD. This glycoprotein quartet is thought to drive membrane fusion through a tightly regulated sequential activation process. Although PrV and HSV share many features and employ the same set of glycoproteins for entry, they show remarkable differences in the requirements for fusion. Whereas HSV strictly depends on all four components, PrV can mediate cell-cell fusion without gD. Moreover, in contrast to HSV, gL-negative PrV provides a unique opportunity for reversion analysis by serial cell culture passaging due to its limited cell-cell spread capacity. By analyzing the modified glycoproteins derived from the gL-negative but infectious revertants as well as by generating chimeric PrV/HSV proteins we are trying to uncover mechanistic details of the fusion process and answer some of the intriguing questions such as: Why is HSV, in contrast to PrV, reliant on gD for membrane fusion and how is PrV able to bypass the requirement for gD? Why do PrV and HSV require so many proteins for entry and how do they interact? Can we reduce the required components and what are the minimal structures? Recent data on these questions will be presented.
Oral 7. Generation of reporter expressing New-World Arenaviruses: a systematic comparison
Lucie Fénéant1, Anne Leske1, Allison Groseth1
1 Junior Research Group - Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems
New World Arenaviruses are bi-segmented negative-sense single-stranded RNA viruses and many, including Junìn virus (JUNV), cause severe hemorrhagic and neurological disease in humans. In contrast, Tacaribe virus (TCRV), a closely related species, is nonpathogenic. To date little is known about the basis for this difference and tools are still needed to directly compare infection with these viruses. In particular, the availability of replication-competent reporter viruses would be a tremendous advantage for applications such as high-throughput siRNA screening, viral particle dynamics in live cell microscopy as well as in vivo virus tracking to investigate key differences between JUNV and TCRV. In a first approach, we have generated tri-segmented TCRV reporter viruses containing two S segments, one in which the glycoprotein (GPC) or nucleoprotein (NP) ORF has been replaced by a reporter gene encoding GFP, mCherry or nanoluciferase, and one containing only the missing viral ORF. In a second approach, we have generated C-terminal reporter fusion proteins for all four TCRV proteins (the polymerase (L), NP, matrix protein (Z) and GPC) either with or without a T2A self-cleavage sequence inserted between the viral protein and the reporter. The functionality of
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these constructs has been evaluated in vitro using minigenome assays, budding assays and/or a cell-cell fusion assays, before transferring successful constructs into the full-length clone system for virus rescue. Thus far, while tri-segmented reporter viruses can be successfully rescued, they are strongly attenuated. NP- and Z- reporter constructs appear to be well tolerated in vitro but still need to be fully evaluated in the viral context. GPC constructs are still being evaluated in vitro, while C-terminal L tagging appears to be unsuitable. These studies will help us to choose viruses with the least attenuation and/or best reporter expression for specific applications and will provide the basis for generating similar JUNV constructs.
Oral 8. Functional investigation of endogenous flaviviral elements in Aedes mosquitoes
Yasutsugu Suzuki*1, Artem Baidaliuk*2, Lionel Frangeul1, Anna B. Crist2, Daisuke Kobayashi3, Hervé Blanc1, Haruhiko Isawa3, Louis Lambrechts2 and Maria-Carla Saleh1
*Equal contribution
1 Institut Pasteur, Viruses and RNA Interference Unit, CNRS Unité Mixte de Recherche 3569, Paris, France. 2 Institut Pasteur, Insect-Virus Interactions Unit, Institut Pasteur, UMR2000, CNRS, 75015 Paris, France. 3 Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo, Japan.
Recent advances in bioinformatics analysis of the genomes of two main mosquito vectors, Aedes (Ae.) aegypti and Ae. albopictus, found a number of integrated viral sequences termed endogenous viral elements (EVEs). One major group of EVEs are closely related to flaviviruses, the genus to which dengue virus and Zika virus belong. In mammals, EVEs regulate closely related exogenous viral infections; however, in mosquitoes the function of EVEs remains unclear. The vast majority of bioinformatically predicted endogenous flaviviral elements are mostly similar to insect specific flaviviruses (ISFs). As such, we have recently identified new EVEs closely related to CFAV and AEFV, both of which are ISFs in Ae. aegypti from Thailand and Ae. albopictus from Japan, respectively. Deep sequencing revealed that EVEs produced primary piRNAs in antisense orientation predominantly in the mosquito ovaries. Initial results using CRISPR/Cas9-based CFAV-EVE knockout mosquitoes showed that CFAV infection is limited in EVE-positive mosquitoes compared to EVE-negative individuals, particularly in the ovaries. Our results suggest mosquito’s EVEs play a specific role in controlling viral replication in ovaries and therefore during vertical transmission of viruses.
Oral 9. Molecular Interactions of HCV core and the long interspersed nuclear element 1 (LINE1) ORF1 protein at HCV assembly sites
Anja Schöbel1, Gerald Schumann2, Eva Herker1
1 Institute of Virology, Philipps University Marburg, Marburg, Germany 2 Paul-Ehrlich-Institute, Langen, Germany.
The Long Interspersed Nuclear Element-1 (LINE1) belongs to the family of retrotransposons and is the only retroelement that is still active in the human genome. It encodes for three proteins: the recently described L1ORF0, the RNA-binding protein L1ORF1p and L1ORF2p, a protein with reverse transcriptase and endonuclease-function. Using a proteomic approach, we identified L1ORF1p to be enriched in lipid droplet (LD) fractions of Hepatitis C Virus (HCV)-infected hepatoma cells. As LDs are described as HCV assembly sites, we investigated a possible interaction between LINE1 and HCV with focus on L1ORF1p. Western blot analysis revealed a slight increase of L1ORF1p protein levels in HCV-infected Huh7.5 cells and confirmed the strong enrichment of L1ORF1p in lipid-rich fractions. In line, immunofluorescence microscopy of HCV-infected Huh7 cells overexpressing HA-L1ORF1p demonstrated close proximity of L1ORF1p to LDs. HCV RNA replication alone did not cause this redistribution, whereas individual expression of HCV core but not HCV NS5A induced the enrichment of L1ORF1p in LD fractions. Together with L1ORF1p, its interaction partners MOV-10 and PABPC1 re-localized to LDs. Concomitantly, all three proteins were found to co-precipitate with HCV core in an RNA-dependent manner, whereas no interaction
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with HCV NS5A was detectable. Further, the HCV RNA genome was enriched in HA-L1ORF1p-precipitated samples. Thus, we conclude that HCV core is part of a ribonucleoprotein particle (RNP) and redistributes it to HCV assembly sites. Overexpression of L1ORF1p did not affect HCV RNA replication; however, if viral spreading is affected is currently under investigation. Using an EGFP-based reporter assay, we studied the impact of HCV infection on LINE1 retrotransposition. Here, we observed a lower retrotransposition frequency in HCV-infected cells, indicating that HCV infection negatively affects LINE1 activity.
Oral 10. Nipah virus matrix protein critically determines inclusion body formation
Nico Becker, Marc Ringel, Laura Behner, Anja Heiner, Andrea Maisner
Institute of Virology, Philipps University Marburg, Marburg, Germany
Nipah virus (NiV) is a zoonotic, BSL-4 classified paramyxovirus that causes respiratory or encephalitic diseases. A hallmark of infections with non-segmented negative-strand RNA viruses such as NiV, is the formation of cytoplasmic inclusion bodies (IBs). Using different imaging techniques, we were able to identify a novel type of IBs at the plasma membrane. Thus, NiV differs from mononegaviruses of the Rhabdo-, Filo- and Pneumoviridae families by forming two IB populations with different formation kinetics and protein compositions (Ringel et al., 2019). IBs in the perinuclear region (IBperi) form rapidly upon expression of the viral nucleocapsid proteins, and are associated with cellular y-tubulin. IBperi can recruit unrelated overexpressed cytosolic proteins but appear not to represent major sites of viral RNA synthesis. This suggests that IBperi represent some aggresome-like compartments. In addition to IBperi, a second IB population which likely represents the site of NiV assembly and budding, is independently formed at the plasma membrane. These so-called IB-PM require the expression of a functional viral matrix (M) protein. M is the central organizer of virus assembly at the plasma membrane and mediates the incorporation of RNPs into budding viral particles by binding to the NiV N protein. As M-N interaction is central for the formation of IB-PM and the generation of NiV particles, we wanted to define the binding regions in the M protein. Using a broad mutagenesis approach (alanine scan), we identified a mutant (M 82-87A) which could still traffic to the cell surface but no longer supported the formation of IB-PM. The mutant M was also unable to recruit N proteins to the plasma membrane suggesting that residues 82-87 (KRKRIR) are part of the N binding site in the NiV M protein. To substantiate this idea in a full viral context, we generated a recombinant NiV encoding the mutant M protein. No IB-PM formation was observed in infections with rNiV-M 82-87A and hardly any infectious NiV particles were released from infected cells. These findings support the idea that the identified 82-87 motif in the NiV M protein plays a crucial role in M-N binding and IB-PM formation, and thus in virus assembly and budding.
Oral 11. Membrane assembly of large DNA viruses; electron microscopy leads the way
Jacomine Krijnse Locker
Unit for service and technology in ultra-structural imaging, center for resources and research in technology, Institut Pasteur, 28, rue du Dr. Roux, 75015 PARIS
For more than 20 years, we have been studying the assembly of the large DNA viruses of the poxvirus family using a variety of imaging techniques. A major controversy in the field is the way these viruses acquire their membrane. Based on classical thin section electron microscopy (EM) carried out in the 1960th it was proposed that the poxvirus vaccinia virus (VACV) acquires a single membrane made de novo in the cytoplasm (Dales and Mosbach, 1968). As other enveloped viruses acquire their membrane from the host by budding at cellular membranes this proposal has been highly criticized.
In 2009 we applied cryo-EM and 3D-EM methods and proposed another unconventional model; VACV recruits membrane derived from the endoplasmic reticulum (ER) that are ruptured. The open intermediates build an open membrane sphere of roughly 350 nm in diameter, shaped by the viral scaffold protein assembled on the convex side of the sphere. The membrane closes after DNA uptake followed by particle maturation (Chlanda et al., 2009; Krijnse Locker et al., 2013). This unconventional model has been criticized as well; molecular mechanisms in favor of one versus other models are, however, missing.
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I will show that members of the giant DNA virus-family, to which VACV belongs, use a similar mechanism of assembly building an open membrane sphere from open intermediates, shaped by an external capsid/scaffold (Suarez et al., 2015; Suarez et al., 2013).
Preliminary result obtained with VACV show a role for both specific cellular lipids as well as viral proteins, some of these preliminary data will be presented.
References
Chlanda, P., Carbajal, M.A., Cyrklaff, M., Griffiths, G., and Krijnse Locker, J. (2009). Membrane rupture generates single open membrane sheets during vaccinia virus assembly. Cell Host & Microbe 6, 81-90. Dales, S., and Mosbach, E.H. (1968). Vaccinia as a model for membrane biogenesis. Virology 35, 564-583. Krijnse Locker, J., Chlanda, P., Sachenheimer, T., and Brugger, B. (2013). poxvirus membrane biogenesis: rupture not disruption. Cell Microb 15, 190-199. Suarez, C., Andres, G., Kolovou, A., Hoppe, S., Salas, M.L., Walther, P., and Krijnse Locker, J. (2015). African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum. Cell Microbiol 17, 1683-1698. Suarez, C., Welsch, S., Chlanda, P., Hagen, W., Hoppe, S., Kolovou, A., Pagnier, I., Raoult, D., and Krijnse Locker, J. (2013). Open membranes are the precursors for assembly of large DNA viruses. Cellular microbiology 15.
Oral 12. Identification of Annexin A3- and Perilipin 2-interacting proteins in hepatitis C virus-infected cells using the proximity labeling methods BioID2 and APEX2
Hanna Bley1, Christoph Krisp2, Hartmut Schlüter2, and Eva Herker1
1 Institute of Virology, Philipps University Marburg, Marburg, Germany 2 Core Facility Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
The hepatitis C virus (HCV) life cycle is tightly connected to the host lipid metabolism with lipid droplets (LDs) serving as an assembly site for HCV. Perilipin 2 (PLIN2), the major LD-decorating protein in hepatocytes, has been shown to play an important role in HCV morphogenesis, as it is required for the formation of infectious HCV particles. Previous studies identified annexin A3 (ANXA3) as an important host factor for HCV. In HCV-infected cells ANXA3 is highly enriched at LDs and is required for HCV particle maturation and egress. In detail, ANXA3 is essential for interaction of the viral envelope protein E2 with the apolipoprotein (Apo) E and for relocalization of ApoE in HCV-infected cells. However, a direct interaction between ANXA3 and viral particles or ApoE was not detected and the molecular details of ANXA3 recruitment to LDs remain unclear. Most likely adaptor proteins mediate the binding and re-routing of ApoE as well as the trafficking of ANXA3 to lipid droplets. To further characterize molecular details of HCV assembly, maturation, and egress and to identify interaction partners within the LD proteome, we used the recently described proximity labeling methods biotin identification BioID2 as well as the engineered ascorbate peroxidase APEX2. Both, BioID2 and APEX2 are promiscuous biotin labeling methods for live cell proteomics to identify physiologically relevant protein-protein interactions in intact cells. BioID2 and APEX2 were genetically fused to ANXA3 or PLIN2 to explore interacting proteins during HCV infection. Therefore, HCV-infected and uninfected cells stably expressing the fusion proteins ANXA3 or PLIN2 were prepared for SILAC labeling to identify differences in binding partners. Immunofluorescence and western blot analysis were used to verify the correct localization of the fusion protein and to detect a pool of biotinylated proteins located in close proximity to the fusion protein. Biotinylated proteins identified by mass spectrometry were investigated for their possible roles in the HCV life cycle. Thus, both methods enable us to study the molecular mechanisms of the HCV life cycle in more detail and will help to decipher the later steps of the HCV viral life cycle.
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Oral 13. The effects of lipoprotein receptors and lipid-lowering drugs on the HCV replication cycle
Francisco Zapatero
The hepatitis C virus (HCV) replication cycle is tightly dependent on the host lipid metabolism. Initial cell entry is mediated by lipoprotein receptors (SR-B1, LDLr) that both bind to apolipoproteins and regulate cellular cholesterol uptake. Subsequently, viruses actively replicate on induced lipidic organelle compartments (membranous web). Finally, HCV particles are assembled by a process that highly resembles VLDL biogenesis. Therefore, molecules that alter the overall cellular lipid homeostasis may drastically affect the HCV replication cycle. We genetically ablated lipoprotein receptors and showed that whereas glycoprotein-mediated HCV entry (HCVpp) was unaltered, HCV cell culture-derived (HCVcc) infection was affected. Strikingly, the inhibitory effect was either mild or unappreciable for single SR-B1 and LDLr knock-out (KO) cell lines but robust for double KO cell lines, suggesting a redundant role of these proteins in the HCV replication cycle. We could also showed that HCV inhibition was not linked to downregulation of cell surface cell entry factors (CD81, Claudin-1, Occludin, NPC1L1) or cholesterol depletion in the KO cells. Alternatively, we disturbed lipid homeostasis by treatment of parental and KO cell lines with known lipid-lowering drugs and observed similar results as for KO cells. Although HCVpp entry was unaffected by all drugs tested, HCVcc infection was inhibited by statins and CETP-inhibitors. Interestingly, statins inhibition was only observed on the KO cell lines, suggesting a compensatory mechanism playing a role in wt cells. In sum, we have shown that lipid metabolism plays a crucial role in the HCV replication cycle. Moreover, we suggest that certain lipid-lowering drugs may exert an antiviral effect on HCV infection.
Oral 14. Unravelling the activities of Junín virus nucleoprotein isoforms in the viral life cycle
Linus Bostedt1, Allison Groseth1
1 Junior Research Group - Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems
The small genome size and limited coding capacity of many RNA viruses necessitates strategies to increase protein diversity at the transcriptional, translational and/or post-translational levels. An example of this approach is Junín virus (JUNV), where the nucleoprotein (NP; 63kD) produces three additional C-terminal isoforms of 53kD, 47kD and 40kD. Using a combined mutagenesis and alanine scanning approach, we could show that the 53kD isoform is the product of alternative translation occurring preferentially from position M80. In contrast, the shorter 47kD and 40kD variants are generated through caspase cleavage. However, while the cleavage sites involved have been mapped, the caspases responsible for cleavage remain unknown. To address this, we are now using purified recombinant NP in an in vitro cleavage assay to identify the relevant caspases. Further, while we are making progress understanding their biogenesis, we still know little about the role of these alternative NP products in the viral life cycle. Therefore, we are, now examining the effects of either these shorter NP isoforms, or full-length NPs that can no longer produce them, using a range of in vitro assays. Using a minigenome assay, we could already show that none of these three shorter NP variants have a role in either supporting or inhibiting viral RNA synthesis. Confirming these results, immunofluorescence assays showed a loss of localization within inclusion bodies, which are the major sites of viral RNA synthesis. Instead, distribution throughout the cytoplasm could be observed, with the smallest isoform (40kD) also localizing in the nucleus. We are now focusing our further analyses on the functions of NP in antagonizing NFkB activation and interferon production. These results illuminate viral strategies that exist to increase protein diversity and functionality, and thereby contribute to a better understanding of the fundamental biology of arenaviruses.
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Oral 15. Quantitative proteomics of Uukuniemi virus-host cell interactions reveals GBF1 as proviral host factor for phleboviruses
Zina M. Uckeley1#, Rebecca Moeller2#, Lars I. Kühn3, Emma Nilsson4, Claudia Robens1, Lisa Lasswitz2, Richard Lindqvist4, Annasara Lenman2, Vania Passos2,5, Yannik Voß1, Christian Sommerauer1, Martin Kampmann1, Christine Goffinet2,6, Felix Meissner3, Anna K. Överby4, Pierre-Yves Lozach1,7,*, Gisa Gerold2,8,*
1 CellNetworks Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany 2 Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany 3 Experimental Systems Immunology, Max Planck Institute of Biochemistry, Martinsried, Germany 4 Division of Virology, Department of Clinical Microbiology, and Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden 5 Instituto De Ciências Biomédicas Abel Salazar, Universidade Do Porto, Porto, Portugal 6 Institute of Virology, Charité, Universitätsmedizin Berlin, Berlin, Germany and Berlin Institute of Health (BIH), Berlin, Germany 7 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, 50 Av. Tony Garnier, 69007 Lyon, France 8 Department of Clinical Microbiology, Virology & Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umea, Sweden
# these authors contributed equally, *: corresponding authors
Highly pathogenic phleboviruses, many closely related to Uukuniemi virus (UUKV), have recently emerged in different parts of the world, posing a global threat for public health. Yet, only few host factors involved in assembly, budding, and exit of these viruses have been studied. Using a high-resolution, label-free mass spectrometry approach, we found that golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) interacts with the UUKV envelope glycoproteins. Both, Golgicide A-mediated inhibition and siRNA-mediated silencing of GBF1 impaired UUKV infection. In depth analysis of the viral infection cycle revealed GBF1 to be involved in UUKV replication, assembly, and release. We finally demonstrated that GBF1 also acts as a proviral host factor for the phleboviruses Toscana virus and Rift Valley fever virus as well as for a broad range of other RNA viruses.
Oral 16. The Ebola virus nucleoprotein NP recruits the nuclear RNA export factor NXF1 for viral mRNA export from inclusion bodies
Lisa Wendt1, Janine Brandt1, Thomas Hoenen1
1 Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems
Ebola virus (EBOV) is an emerging negative sense RNA virus that causes severe outbreaks of viral hemorrhagic fever in humans. There is only limited knowledge regarding the interactions of this virus with cellular host factors during its life cycle. Using a genome-wide siRNA screen we have recently identified the nuclear RNA export factor 1 (NXF1) as important for the EBOV life cycle. NXF1 is a crucial component of the cellular mRNA export pathway, which is accessed by many viruses replicating in the nucleus. However, a possible mode of interaction between NXF1 and the cytoplasmically replicating EBOV and NXF1’s function in the EBOV life cycle are completely unknown. To address these questions, we performed co-immunoprecipitation (CoIP) assays of NXF1 with several EBOV proteins, followed by subsequent deletional mutagenesis studies. These studies revealed an interaction of NXF1 with the EBOV nucleoprotein NP. Interestingly, NP and cellular RNAs seem to compete for binding of the RNA-binding domain of NXF1. Colocalization studies showed that an RNA binding-deficient NXF1 accumulates in NP-derived inclusion bodies, which have been shown to be sites of virus genome replication and transcription. Knockdown experiments of NXF1 in the context of minigenome assays combined with strand-specific RT-qPCR to dissect the viral RNA synthesis steps further demonstrated that NXF1 is necessary for viral gene
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expression, but only to a very limited extent for viral genome replication or transcription. RNA-CoIPs showed that NXF1 is capable of interacting with viral mRNA, but not with viral (mini-)genomic RNA. Taken together, our results suggest that NP usurps NXF1 to export viral mRNAs from inclusion bodies and towards host ribosomes. This better understanding of the role of NXF1 during the EBOV life cycle might provide a basis for the development of new therapeutics against EBOV and possibly also other emerging viruses.
Oral 17. Finding the elusive dsRNA receptor involved in antiviral immune response in insects
Fletcher, Sabrina Johanna; Tomé-Poderti, Lorena; Frangeul, Lionel; Saleh, Carla
Institut Pasteur, Paris, France
Several (re)emerging viral infectious diseases, such as Dengue and Zika, are transmitted to humans by insect vectors like mosquitoes. One of the key factors that modulates whether an insect is competent or not to transmit a given pathogen is its innate immune response. RNA interference (RNAi) is the main antiviral response in insects and is triggered by the recognition of viral double-stranded RNA (dsRNA). During the RNAi-immune response, infected cells transmit an antiviral signal to non-infected cells generating an antiviral state. However, the nature of the RNAi-immune signal and how the non-infected cells recognize and internalize this signal remain unsolved. We hypothesize that during viral infection, viral dsRNA is released due to cell lysis and binds a membrane cell receptor/s to prime non-infected cells. Our project aims to identify and characterize the dsRNA cell receptor that binds extracellular dsRNA in insects. Here, I will present a reliable in vitro system developed in the lab to find the receptor in the insect model Drosophila melanogaster. Using an “omics” approach that integrated transcriptomics, proteomics and in silico analysis, the lab identified a list of potential candidates that could fulfill the role for a cell receptor. Among them, several ion and iron transporters are of particular interest. I will present their study and characterization. The discovery of the dsRNA cell surface receptor could result in the development of new strategies to control the spread of vector-borne viral diseases by manipulating the insect vector.
Oral 18. Analysis of determinants controlling antiviral activity of defective interfering influenza A virus RNAs
Prerna Arora1, Sabine Gärtner1, Najat Bdeir1, Martin Schwemmle2, Udo Reichl3, Michael Winkler1, Stefan Pöhlmann1
1 Infection Biology Unit, German Primate Center, Göttingen, Germany 2 Institute of Virology, Medical Center University of Freiburg, Freiburg, Germany 3 Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Magdeburg, Germany
Influenza A virus (IAV) infection is a major source of global morbidity and mortality. The presence of deletions in IAV segments can result in the production of defective interfering particles (DIP). Upon coinfection with influenza A virus DIPs suppress amplification of wt RNAs thus exerting antiviral effects. However, the molecular mechanisms underlying antiviral activity are incompletely understood. DI 244 is a prototype DI RNA derived from genomic segment 1 harboring a large central deletion. We investigated whether the truncated PB2 open reading frame (ORF) of DI 244 and the segment length of the DI RNA impact antiviral activity. Mutations and deletions in IAV segment 1 RNA-encoding plasmids were introduced by PCR-based mutagenesis. A mini-replicon system was employed to study IAV genome replication and its inhibition by DI RNAs. For analysis of antiviral activity of DI RNAs, a co-culture of MDCK and 293T cells was co-transfected with plasmids encoding DI RNAs derived from segment 1 and wt IAV genomic segments 1-8. Pure DIPs were produced in a novel cell culture system employing PB2-expressing 293T and MDCK cells. Antiviral activity of pure DIPs was assessed by co-infection with influenza A/PR/8/34 virus. Mutation of the three start codons within the PB2 ORF revealed that the truncated PB2 protein did not contribute to antiviral activity of DI 244. In contrast, the analysis of segment 1 determinants with
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nested deletions showed that deletion size was inversely correlated with inhibitory activity in the mini-replicon system and similar results were obtained for segments 2 and 3. Thus, in the context of genome replication any IAV segment harboring a deletion might act as DI RNA, with DI RNA length being the major determinant of antiviral activity. Furthermore, co-transfection of plasmids encoding segment 1-derived DI RNAs and wt segments 1 to 8 showed that also in the context of infectious IAV production, DI RNA length is the major determinant of antiviral activity. However, co-infection of DIP supernatants together with A/PR/8/34 virus exhibited modest antiviral activity but not a clear length dependency among the segment 1 determinants. We speculate that interferon (IFN) might play a role in determining antiviral activity. We are currently using an interferon (IFN) competent cell system to analyze the impact of DI RNA length on antiviral activity.
Our results suggest that DI RNAs length is a central determinant of antiviral activity. IFN might play a major role in determining antiviral activity.
Oral 19. Innate immune response to Ebola virus
J. Wildemann1, L. Rivière1, C. Helmer1, T. Hoenen2, R. König1, 3
1 Host-Pathogen Interactions, Paul-Ehrlich-Institut, Langen, Germany; 2 Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald, Germany 3 Immunity and Pathogenesis Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, USA
Introduction: Initial Ebola virus (EBOV) replication occurs in dendritic cells (DCs) and macrophages, the sentinel cells of our immune system. However, infected DCs fail to orchestrate an effective immune response. Virulence is partly associated to EBOV protein VP35, an IFN-antagonist which effectively counteracts the activation of RNA receptors from the RIG-I-like receptor (RLR) pathway family. Therefore, the early immune response to EBOV seems to be crucial for disease outcome.
Objectives: Our goal is to determine pathogenic-associated molecular patterns (PAMPs) of EBOV and to identify the cellular immune sensors and co-regulatory molecules involved in early innate sensing.
Materials & Methods: A transcription- and replication-competent virus-like particle (trVLP) system that allows life cycle modeling of EBOV under BSL1 conditions is used. Innate sensing is measured by monitoring expression of the direct IRF3-target gene ISG54 by qRT-PCR or by type-I IFN ELISA.
Results: We successfully established the trVLP replication system to infect monocyte-derived DCs (MDDCs). Two different VP35 mutants were generated with the aim to block their function as antagonists of the early sensing pathway, but to retain their role in replication and transcription. Production of trVLPs including the VP35 mutants in HEK 293T cells, leads to a strong ISG54 and IFN-β response compared to wt VP35, suggesting that mutant VP35 proteins lost their antagonistic activity compared to wt VP35. Nevertheless, infection of cells with mutant VP35 trVLPs does not lead to an immune response. To understand the lack of innate response to trVLP infection, we first assessed the immunostimulatory potential of naked Ebola RNAs isolated from trVLPs in a quantitative assay using monocyte-derived dendritic cells (MDDCs) as a model for highly immunocompetent cells. Upon transfection of viral nucleic acids into immunocompetent cells, high ISG54 inductions are observed, suggesting that viral RNA components are sensed. To identify the particular innate pathways that are triggered by Ebola RNA, we exposed a panel of THP-1 knockout (KO) cell lines deficient for key molecules of various sensing pathways and known sensors to Ebola RNA. As expected, PMA-THP-1 cells deficient in MAVS lose the ability to trigger an ISG54 response upon stimulation with Ebola RNA, suggesting members of the RLR family as initial sensors. In future, we are planning on testing further THP-1 KO cell lines including KOs of RNA-modifying enzymes, to determine the exact PAMP and PRR pathways initiating the response.
Conclusion: Our study will lead to a better understanding of EBOV-host interactions and early sensing events and might help to find better therapeutic strategies.
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Oral 20. Molecular interactions of tick-borne encephalitis virus proteins and their role in the ER-stress response
Veronika J. M. Breitkopf1,2 and Imke Steffen1,2
1 Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Hannover, Germany 2 Institute for Biochemistry, University of Veterinary Medicine, Hannover, Germany
Keywords: Tick-borne encephalitis virus, flavivirus, viral protein expression, protein modification, protein-protein interaction
Tick-borne encephalitis virus (TBEV) belongs to the genus Flavivirus within the family Flaviviridae and can cause severe neurological complications such as encephalitis, meningitis or meningoencephalitis upon entry of the central nervous system. The mechanisms leading to neuronal loss of function and death are poorly understood, but likely result from a mix of direct viral replication and cellular responses to infection. The TBEV genome encodes a single open reading frame containing ten viral proteins, including three structural proteins – capsid protein C, precursor membrane protein M (prM) and envelope protein E – and seven non-structural proteins – NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The viral proteins are translated into a single polyprotein, which is co- and post-translationally cleaved into individual proteins by viral and host proteases. This complicates the study of individual TBEV proteins and our current understanding of their role in cell damage is incomplete. The coding regions of all 10 TBEV proteins were individually cloned and recombinantly expressed in mammalian cells. Protein expression, modification and localization was analyzed by Western blot and immunocytochemistry. Oligomerization was observed for prM, E, NS1, NS2B, NS3, NS4A and NS5. Co-immunoprecipitation identified C and prM, NS2B and NS3 as well as NS4A and NS4B as hetero-oligomers. Analysis of cell morphologies following expression of individual TBEV proteins showed structural changes in cells expressing the E and NS4A proteins. Moreover, TBEV proteins involved in the induction of the ER stress response caused by ER remodeling in the course of TBEV infection are being identified.
Oral 21. Bad, Noxa and Puma are key regulators of Tacaribe virus-induced apoptosis
Julia Holzerland1, Logan Banadyga2, Allison Groseth1
1 Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald - Isle of Riems, Germany 2 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada
New World arenaviruses represent an important group of zoonotic pathogens that present a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever with neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell mechanisms involved in the response to an infection, or how they contribute to virulence; however, TCRV strongly induces apoptosis in infected cells, whereas JUNV does not. In order to reveal which signaling pathways are responsible for TCRV-induced apoptosis, a variety of methods were used to investigate activation of established apoptotic pathways. We observed a number of canonical features, including mitochondrial disorganization, Cyt c release, PS flipping and caspase activation, confirming involvement of the intrinsic apoptotic pathway. Further, our investigations examined the relevance of apoptosis regulators, such as p53 and PML for initiating apoptotic signaling, as well as the involvement of pro-apoptotic BH3-only proteins. In particular, the pro-apoptotic BH3-only proteins Noxa and Puma, both p53-regulated genes, were strongly upregulated. Interestingly, TCRV infection also increased expression of another pro-apoptotic BH3-only protein, Bad, although this was mostly found in its inactive form as Phospho-Bad. These data clearly suggest that the pro-/anti-apoptotic balance in TCRV infected cells is controlled at least in part by virus-induced regulation of Bad, Noxa and Puma. Consistent with this model, knockout of Noxa and Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad resulted in increased apoptotic activity. These findings confirm a role of these BH3-only factors in the sensing of TCRV-induced cell stress and provided us with a more detailed understanding regarding the role of the apoptotic machinery in response to arenavirus infection.
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Poster presentations
Poster 1. Identification of optimal strategies for the rescue of non-attenuated tag- or reporter-expressing Ebola viruses
Bianca S. Bodmer1, Thomas Hoenen1
1Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems
Ebola virus (EBOV) causes hemorrhagic fever in humans and nonhuman primates with case fatality rates of 40-60%. Working with infectious EBOV is restricted to biosafety level (BSL) 4 laboratories, but several life cycle modelling systems have been developed to mimic different aspects of the filovirus life cycle under BSL1 or 2 conditions. In the past, recombinant EBOVs that express different reporters, such as luciferases or green fluorescent protein (GFP) from additional transcription units or as fusion proteins, have been rescued. These viruses are important tools for the study of EBOV, and their use includes enhanced detection and quantification for high throughput screening approaches, analysis of intercellular localization and tissue distribution, and the evaluation of pathogenesis in vivo. However, they all show, at least in vivo, high attenuation compared to wild type virus. To address this issue by finding the best options for addition of tags or reporter proteins to different EBOV proteins, we fused a flag-HA tag or GFP at different sites of the EBOV proteins NP, VP35, VP30, VP40, and L, and analyzed the tolerability of these additions with respect to protein function. Furthermore, GFP was fused to the C terminus of proteins that best tolerated C-terminal tagging using a T2A site, which results in translation of two open reading frames from the same mRNA. For assessment of a possible impact on protein function we analyzed the tagged proteins in various life cycle modelling systems. Interestingly, we observed a wide spectrum of tolerance at different sites and with respect to different protein functions, ranging from no changes in protein function up to more than 97% loss of function. The obtained data will be used to rescue selected recombinant EBOVs encoding reporter proteins or molecular tags at sites with low impact on functionality of the respective tagged protein, and thus minimal attenuation in vitro and in vivo.
Poster 2. Mapping the interactions of the filovirus matrix protein VP40 with nucleocapsids using chimeric tetracistronic transcription- and replication-competent virus-like particles
Janine Brandt1, Stefanie Braun1, Thomas Hoenen1
1 Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald Isle of Riems, Germany
Filoviruses (which include the genera of ebola-, marburg- and cuevaviruses) are zoonotic pathogens causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and scope of filovirus outbreaks has increased, highlighting the importance of better understanding the molecular interactions of filoviral proteins in order to be able to better combat these viruses. The filovirus matrix protein VP40 fulfills a crucial role in the virus lifecycle, mediating assembly and budding of virions from infected cells. During this process, nucleocapsids, consisting of the filoviral RNA genome and the proteins NP, L, VP35 and VP30, are recruited into virus particles by VP40. Previous studies have shown that the ebolavirus matrix protein VP40 is not able to interact with marburgvirus nucleocapsids and vice versa, suggesting that nucleocapsid proteins and VP40 have to be from the same filovirus genus. In this study we investigated whether VP40 and nucleocapsid proteins also have to be from the same species. To address this question we used chimeric tetracistronic transcription- and replication-competent virus-like particle (trVLP) systems featuring VP40 genes from different ebolavirus and cuevavirus species. Interestingly, our results suggest that for nucleocapsid interaction VP40 is exchangeable within but not between the filovirus genera. In order to further map the interaction site of VP40 with the nucleocapsid proteins we generated tetracistronic minigenomes with chimeric ebolavirus/cuevavirus VP40 genes. By using this approach, we could show that most of the C-terminal part of VP40 is not involved in the interaction with nucleocapsids, whereas the N-terminal part of VP40 seems to be important for nucleocapsid recruitment. Mapping the interaction site of VP40 with nucleocapsids will not only increase our
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understanding of the filovirus lifecycle, but also help to define novel therapeutic targets against these viruses.
Poster 3. Establishment of RT-PCR assays for the analysis of the virus-induced immune response in M. nataliensis
Nele Burckhardt
Lassa virus (LASV) is a negative-sense RNA virus belonging to the Arenaviridae family1. It is the cause of a haemorrhagic fever called Lassa fever (LASF), which affects around 150,000-300,000 people annually and causes up to 5,000 deaths per year2. LASF is endemic in West African countries such as Sierra Leone3, Liberia4, Guinea5 and Nigeria6. The main reservoir host of LASV is the commensal Multimammate rat Mastomys natalensis7, which often lives close to human households8. Since Mastomys shed virus in their urine, the transmission to humans mainly occurs through contact with rodent excreta and contaminated food or due to direct contact with infected animals3,9. Furthermore, nosocomial human-to-human transmission has been observed7. In order to precisely predict LASF outbreaks, further research on the virus-host interactions of LASV as well as related arenaviruses and their natural host is needed. Previous experiments have shown that certain LASV strains are able to cause chronic infections in Mastomys natalensis whereas others are not. A possible cause for host-species barriers are differences in the antiviral immune response against the various LASV strains. The Interferon system plays a crucial role in the antiviral defence of the host organism and Interferon-stimulated genes (ISG) are able to target almost any step during the viral life cycle11 The major focus of this project is to characterize the host immune response to examine the underlying mechanisms causing these host restrictions. We aim to establish RT-PCR-based assays to detect changes in ISG expression of Mastomys-derived cells after stimulation with viral proteins from different LASV strains. Furthermore, infection experiments with non-pathogenic, LASV-related viruses will be conducted in order to compare the ability of the antiviral response of the host cells.
Poster 4. Hepatitis E virus trafficking in polarized stem cell-derived hepatocyte-like systems
Charlotte C. Decker1, 2, Andrew Freistaedter1 and Viet Loan Dao Thi1, *
1 Schaller Research group at Department of Infectious Diseases and Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany 2 Heidelberg Biosciences International Graduate School, Heidelberg University, 69120 Heidelberg, Germany * Correspondence: [email protected]
Hepatitis E virus (HEV) is the major cause of acute hepatitis in the world. HEV is principally transmitted through the fecal-oral route. The virus enters via the gastrointestinal tract and infects the liver, where it enters polarized hepatocytes from the bloodstream; viral progenies are then released differentially from the hepatocyte’s membrane, quasi-enveloped HEV particles predominantly from the basolateral side and non-enveloped HEV particles from the apical side. We previously developed a novel differentiation protocol that allows columnar polarization of stem cell-derived hepatocyte-like cells (HLCs) in Transwells. We will use this novel system to identify and characterize polarized trafficking and secretion routes of HEV. The HEV genome contains three open reading frames (ORFs). ORF1 encodes the domains responsible for genome replication, ORF2 codes for the capsid protein, and ORF3 for a small phosphoprotein that is critical for viral egress. SNAP-tag is a self-labeling enzyme that enables pulse chase labeling with live cell-compatible dyes. We cloned the ORF3 protein with the SNAP-tag at its N-terminus (SNAP-ORF3) into a doxycycline-inducible lentiviral expression system. First, we transduced a hepatoma cell line with SNAP-ORF3 and confirmed co-localization of SNAP and ORF3 by confocal microscopy. We also verified SNAP-ORF3 co-localization with multivesicular body marker CD63, in agreement with previous reports. Then, we transduced the intestinal cell line Caco-2, which can be polarized when grown on Transwells, with SNAP-ORF3 and observed an accumulation at the apical membrane. We are now optimizing the transduction of polarized HLCs with SNAP-ORF3. We are further optimizing the
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pulse-chase labelling and imaging conditions together with testing different live cell-compatible markers of subcellular compartments. Then, we will proceed with labelling ORF3 in live polarized HLCs and apply quantitative single particle tracking analysis. In the next step, we will trans-complement the cells with the other viral factors by expressing tagged ORF2 and ORF3 in stem cells and providing a HEV replicon encoding the HEV replicase in trans. Our efforts should lead to a better understanding of HEV secretion but also of the fine-tuned spatio-temporal dynamics and regulations of the polarized trafficking machinery in hepatocytes in general.
Poster 5. Virus-encoded amyloidogenic protein
Psylvia Léger1,2, Eliana Nachman3, Karsten Richter4, Carole Tamietti5, Robin Burk2, Susann Kummer2, Megan Stanifer2,4,7, Michèle Bouloy8, Steeve Boulant2,4,7, Hans-Georg Kräusslich2, Xavier Montagutelli6, Marie Flamand5, Carmen Nussbaum-Krammer3, Pierre-Yves Lozach1,2,9,*
1 CellNetworks – Cluster of Excellence, University Hospital Heidelberg, Heidelberg, Germany 2 Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany 3 Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany 4 DKFZ, Heidelberg, Germany 5 Structural Virology, Institut Pasteur, Paris, France 6 Mouse Genetics Laboratory, Institut Pasteur, Paris, France 7 Schaller research group at CellNetworks, Heidelberg, Germany 8 Unité de Génétique Moléculaire des Bunyavirus, Institut Pasteur, Paris, France 9 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, Lyon, France
Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such Alzheimer’s and Creutzfeldt-Jakob disease. Investigating Rift Valley fever virus, an important human pathogen, we found that its major virulence factor, the protein NSs, forms filamentous structures in the brain of infected mice. NSs assembled into nuclear and cytosolic disulfide bond-dependent, amyloid-like fibrillary aggregates in infected cells. NSs structural arrangements exhibited characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils could be visualized in real-time. They did not require any artificial activation and grew in an amyloid-fashion within 5 hours. Our study provides the first demonstration that viruses can encode amyloidogenic fibril-forming proteins. This finding has strong implications for future research on amyloid aggregation and toxicity in general.
Poster 6. Deciphering the pathway of Toscana virus induced apoptosis
Felix Streicher1, Franziska Wölfl1, Psylvia Léger1, Shawon Gupta1, Pierre-Yves Lozach1,2
1 CellNetworks – Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany 2 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, Lyon, France
Several members of the Phlebovirus genus were identified to cause mild to severe diseases in human and cattle. One such representative is Toscana Virus (TOSV), which has a neurovirulent characteristic causing meningitis and encephalitis in infected patients. TOSV was previously described as one of the most prevalent arthropod-borne viruses in Europe and is thus of special interest for investigation. Previous studies in a mouse model suggest the neuropathogenic capacity of the virus, which is essential for causing severe forms of disease, being based on apoptotic degeneration of neurons. This marks the viral induction of regulated cell death as an important part of TOSV pathogenicity. Therefore, this project aims to characterize cell death pathways triggered in human cells upon infection with TOSV. A special focus will be set on apoptotic pathways of regulated cell death, since preliminary live cell imaging of TOSV infected human cells suggested caspase-3 dependent apoptosis as the triggered cell death mechanism. Shedding light onto the cell death pathways induced by infection with TOSV could potentially lead to novel strategies against severe forms of neuropathogenic TOSV infection and might indicate potential target points for also other related pathogenic viruses.
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Poster 7. Lipid droplet biogenesis regulates HCV RNA replication
Isabelle Reichert, Gabrielle Vieyres, Thomas Pietschmann
By serving as a triacylglycerol reservoir, lipid droplets are the main energy source of cells but also provide structural lipids for membrane formation and remodeling. Hepatitis C (HCV) virus hijacks these lipid droplets for its own replication, which results in the formation of the characteristic infectious lipo-viro-particle. However, the interplay of HCV and the lipid compartment is not fully understood yet. It has been shown earlier that DGAT1, a cellular transmembrane enzyme catalyzing the conversion of diacylglycerols to triacylglycerols and involved in lipid droplet biogenesis, is crucial for the assembly of HCV lipo-viro-particle. However, in recently performed overexpression experiments in our lab we observed an inhibiting effect of DGAT2 on HCV replication, which catalyzes the same enzymatic reaction as DGAT1.
In my recently started PhD project I want to examine which replication step of HCV is affected by DGAT2 and, further, investigate the mechanism of DGAT2 inhibition of HCV replication. Therefore, I am currently testing the effect of both DGAT1 and DGAT2 on HCV RNA translation. This is done by using a dual luciferase reporter system that allows the quantitative comparison of HCV IRES-mediated in respect to cap-mediated translation within control vs. DGAT1- or DGAT2-overexpressing cells. Future experiments will address the precise viral and cellular determinants of DGAT2 antiviral effect and unravel the mechanisms behind the opposite effects of DGAT1 and DGAT2 on HCV infection.
Poster 8. Cell biology of Germiston virus entry
Stefan Windhaber1, Jana Koch1, Pierre-Yves Lozach1,2
1 CellNetworks Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany 2 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, 50 Av. Tony Garnier, 69007 Lyon, France
Germiston virus (GERV) is a mosquito-borne orthobunyavirus endemic in southern Africa that causes mild febrile disease in humans which, unlike many other orthobunyaviruses, can be studied under BSL 2 conditions. The genus Orthobunyavirus in the order of the Bunyavirales includes many viruses that cause febrile diseases or even meningoencephalitis in humans. The closely related Oropouche virus, which is endemic in South and Central America, is a frequent cause of arboviral febrile disease in Brazil and can also cause meningoencephalitis in severe cases. Also, it can only be studied under BSL 3 conditions. Currently, there are no specific antiviral treatments or vaccinations available for most of these viruses. As only little is known about the infection of orthobunyaviruses at a cellular and molecular level, we aim to investigate early GERV-host cell interactions which might help to identify potential targets for the treatment of GERV. Using flow cytometry- and microscopy-based approaches, we will examine the different GERV entry steps, as virus binding, internalization, intracellular trafficking, and fusion into the cytosol. Furthermore, we are elucidating the structural organization of GERV particles using a combination of fluorescence and cryo-electron microscopy. My preliminary experiments show that GERV can be produced from BHK-21 cells to very high titers laying the groundwork for many of the upcoming assays.
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Poster 9. Saving private Toscana
Franziska Wölfl1, Nadia Oreshkova2, Felix Pahmeier1, Paul Wichgers Schreur2, Jana Koch1, Megan Stanifer1, Zina Maria Uckeley1, Steeve Boulant1, Psylvia Léger1, Jeroen Kortekaas2, Pierre-Yves Lozach1
1 CellNetworks Cluster of Excellence and Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany 2 Laboratory of Virology, Wageningen University, 6701 Wageningen, The Netherlands 3 IVPC UMR754, INRA, Univ. Lyon, Université Claude Bernard Lyon 1, EPHE, 50 Av. Tony Garnier, 69007 Lyon, France
Toscana virus (TOSV, Phenuiviridae, Phlebovirus), is largely distributed over the Mediterranean basin. It was first isolated in Italy from phlebotomine sandflies in 1971. In humans, TOSV causes febrile illness, which can evolve to meningoencephalitis in severe cases. Despite a constant expansion in the region, TOSV remains poorly characterized at the molecular level. Mutagenesis-based studies are however a prerequisite to better understand the cell biology of TOSV, from both virus-host cell interactions and replication perspectives. Here, we engineered a T7 RNA polymerase-driven reverse genetic system to recover infectious particles of TOSV lineage B strain H4906 (France, 2004). Our results show that rescued virus (rTOSV) forms plaques in titration assays similar to those of our TOSV lab strain and is stable over more than 4 passages. When viral particles were amplified, semi-purified and analyzed by Coomassie, the typical bands corresponding to the virus envelope proteins (GN and GC) and nucleoprotein were observed. Immunoblot against GN and GC, the nucleoprotein N, and the non-structural protein NSs expressed in cells exposed to the virus confirmed the TOSV infection. We then introduced random stop codons in the sequence coding for TOSV NSs and were able to rescue a rTOSV mutant impaired for NSs expression as confirmed by immunoblot. Altogether, our study is the first demonstration that TOSV can be genetically engineered and recovered from plasmid DNAs. Our data therefore opens a new avenue of research on this neglected emerging pathogen that represents a growing threat for human health in the Mediterranean region and neighboring countries.
Poster 10. Hepatitis E virus cell entry
Rebecca Fu1, Andrew Freistaedter1, Lucas Knapp1, and Viet Loan Dao Thi1,*
1 Schaller Research group at Department of Infectious Diseases and Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany * Correspondence: [email protected]
Hepatitis E virus (HEV) is a major causative agent of acute fulminant hepatitis. The virus is mainly transmitted via the oral–fecal route through contaminated drinking water. HEV particles from feces are predominantly found to be non-enveloped, while those circulating in the serum possess a lipid envelope derived from the host. The cell entry routes of both, enveloped (eHEV) and non-enveloped (nHEV) HEV particles, are poorly described. In this study, we attempt to elucidate the mechanism of entry into host cell by nHEV. nHEV has a higher density and is more infectious than eHEV due to the lack of the lipid envelope. Endocytic bypass assays reveal that nHEV entry into host cells is dependent on endocytosis and low pH. We are further characterizing the cell entry routes by biochemical and imaging approaches.
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List of participants and email contact addresses
Bryan Arias [email protected]
Prerna Arora [email protected]
Nico Becker [email protected]
Stephan Becker [email protected]
Lesley Bell-Sakyi [email protected]
Hanna Bley [email protected]
Bianca Bodmer [email protected]
Linus Bostedt [email protected]
Steeve Boulant [email protected]
Janine Brandt [email protected]
Veronika Breitkopf [email protected]
Nele Burckhardt [email protected]
Petr Chlanda [email protected]
Claudia Claus [email protected]
Charlotte Decker [email protected]
Lucie Feneant [email protected]
Sabrina Fletcher [email protected]
Gisa Gerold [email protected]
Allison Groseth [email protected]
Eva Herker [email protected]
Thomas Hoenen [email protected]
Julia Holzerland [email protected]
Sandra Junglen [email protected]
Franziska Kaiser [email protected]
Jared Kirui [email protected]
Steffen Klein [email protected]
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Hannah Kleine-Weber [email protected]
Jana Koch [email protected]
Cassandra Koh [email protected]
Alain Kohl [email protected]
Psylvia Léger [email protected]
Jacomine Krijnse-Locker [email protected]
Pierre-Yves Lozach [email protected]
Karen Mossman [email protected]
Fu Rebecca [email protected]
Isabelle Reichert [email protected]
Anja Schöbel [email protected]
Sebastian Sprengel [email protected]
Felix Streicher [email protected]
Yasutsugu Suzuki [email protected]
Zina Uckeley [email protected]
Melina Vallbracht [email protected]
Lisa Wendt [email protected]
Johanna Wildemann [email protected]
Benedikt Wimmer [email protected]
Stefan Windhaber [email protected]
Sophie Winter [email protected]
Franziska Wölfl [email protected]
Francisco Zapatero [email protected]
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Notes
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