ITALY ITALY Abstract Book Table of Content Oral Presentations…………………….. Page 3 Poster Presentations………………….. Page 96 Abstract Author Index…………………. Page 336 2 NSV 2018, Verona – Abstract Book ORAL PRESENTATIONS BREAKING AND ENTERING - viral entry Abstract final identifier: 1 HUMAN LEUCOCYTE ANTIGEN DR (HLA-DR) HOMOLOGS ARE CROSS-SPECIES ENTRY RECEPTORS FOR BAT INFLUENZA VIRUSES Silke Stertz* 1, Umut Karakus1, Thiprampai Thamamongood2, Kevin Ciminski2, Wei Ran2, Sira C. Günther1, Davide Eletto1, Benjamin G. Hale1, Adolfo Garcia-Sastre3, Martin Beer4, Martin Schwemmle2 1University of Zurich, Zurich, Switzerland, 2Medical Center University of Freiburg, Freiburg, Germany, 3Icahn School of Medicine at Mount Sinai, New York, United States, 4Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany Abstract: In 2012 and 2013 two novel influenza A viruses were discovered in South American bat species. Remarkably, the hemagglutinin of these bat influenza viruses was found not to bind the canonical influenza virus receptor, sialic acid, or any other glycan, despite high sequence and structural homology with conventional influenza A virus hemagglutinins. Using transcriptomic profiling of susceptible versus non-susceptible cells, in combination with genome-wide CRISPR/Cas9-based screening, we identified the MHC-II complex HLA-DR as proteinaceous receptor for bat influenza viruses. CRISPR/Cas9- mediated knockout of HLA-DR rendered susceptible cells completely resistant to bat influenza but not conventional influenza virus infection, whereas ectopic expression of HLA-DR in non-susceptible cells conferred susceptibility to bat influenza virus infection. Expression of HLA-DR homologs from three different bat species, pigs or chickens also conferred susceptibility. Notably, infection of mice with bat influenza virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant to infection. Collectively, our data identify HLA-DR homologs from multiple species as receptors for bat influenza virus, suggesting the potential for broad vertebrate tropism. 3 BREAKING AND ENTERING - viral entry Abstract final identifier: 2 NECTINS TRANSFER CYTOPLASM BETWEEN CELLS AND CAN SPREAD MEASLES VIRUS TO NEURONS Alex Generous1, Oliver Harrison2, Regina Troyanovsky3, Mathieu Mateo1, Chanakha Navaratnarajah1, Ryan Donohue1, Christian Pfaller1, Alina Sergeeva2, Indrajyoti Indra3, Theresa Thornburg4, Irina Kochektova4, Matthew Taylor4, Sergey Troyanovsky3, Barry Honig2, Lawrence Shapiro2, Roberto Cattaneo* 1 1Molecular Medicine, Mayo Clinic, Rochester MN, 2Biochemistry and Molecular Biophysics, Columbia University, New York, 3Dermatology, Northwestern University, Chicago, 4Microbiology and Immunology, Montana State University, Bozeman, United States Abstract: We discovered a process mediating intercellular transfer of cytoplasmic materials. Cells expressing the adherens junction protein nectin-1 (N1) take in plasma membrane patches and cytoplasmic materials from cells expressing other nectins. This process is most active during cell adhesion, 2-8 hours post-co-culture. It is most efficient from cells expressing nectin-4 (N4) towards cells expressing N1. And, it depends on the N1 cytoplasmic tail: its deletion prevents transfer, while its exchange with the N4 cytoplasmic tail reverses transfer direction. We term the process nectin-elicited cytoplasm transfer (NECT). Nectin family proteins serve as receptors for positive strand RNA viruses like poliovirus, large DNA viruses like herpes simplex virus, and negative strand RNA viruses such as measles virus (MeV). MeV infections can cause subacute sclerosing panencephalitis (SSPE), a rare but lethal disease. Because of SSPE and of other neurological diseases caused by MeV and related animal viruses, a neuronal receptor has been postulated but no consensus candidate has emerged. We show that N4-expressing, MeV-infected epithelial cells transmit infection to axons of N1-expressing primary neurons that cannot be infected by MeV particles. Thus NECT can spread MeV infections to neurons, circumventing the need for a neuronal receptor. We have evidence that NECT functions through an endocytic pathway, and seek to understand how and where MeV ribonucleocapsids escape it. 4 NSV 2018, Verona – Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 3 STRUCTURE OF FULL-LENGTH INFLUENZA HAEMAGGLUTININ Donald J. Benton* 1, Andrea Nans2, Lesley J. Calder2, Davide Corti3, Steven J. Gamblin1, Peter B. Rosenthal2, John J. Skehel1 1Structural Biology of Disease Processes Laboratory, 2Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom, 3-, Humabs BioMed SA, Bellinzona, Switzerland Abstract: Influenza A virus Haemagglutinin (HA) is the surface glycoprotein responsible for receptor binding and membrane fusion. Previous structural characterisation of HA has been limited to a soluble ectodomain which does not contain membrane proximal or transmembrane regions. We present structures, determined by cryo-EM, of full-length detergent- solubilised HA in isolation and complexed with a Fab fragment from an infectivity neutralising, H1 subtype-specific antibody, that recognises the ecto-domain-transmembrane domain junction. The structures of the ectodomain compare favourably in resolution with those previously determined by x-ray crystallography. Our description of the previously undetermined parts of the molecule may be important in relation to the immune recognition of the membrane proximal region of HA in antibody binding and antibody induction. 5 BREAKING AND ENTERING - viral entry Abstract final identifier: 4 STRUCTURAL BASIS OF LOW-DENSITY LIPOPROTEIN RECEPTOR RECOGNITION BY VSV GLYCOPROTEIN Laura Belot1, Nikolic Jovan1, Pierre Legrand2, Hélène Raux1, Yves Gaudin1, Aurelie Albertini* 1 1Institut de Biologie Intégrative de la Cellule, CNRS, 2Synchrotron SOLEIL, GIF SUR YVETTE, France Abstract: Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. VSV G mediates both virus attachment to its receptor and fusion of the viral envelope with the endosomal membrane. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. We demonstrate that VSV G is able to independently bind two distinct cystein-rich (CR) domains (CR2 and CR3) of LDL-R with similar affinities (Kd ~ 5µM). The biological relevance of this interaction was demonstrated by the ability of both CR2 and CR3 to inhibit VSV infection. We obtained two crystal structures of G in its pre-fusion conformation in complex with CR2 and CR3 revealing that the binding sites of CR2 and CR3 on G are identical. CR domains recognition by VSV G involves basic residues pointing toward the calcium-coordinating acidic residues present in each CR. Mutations of two of these residues abolish G ability to bind to LDL-R without impairing G fusion activity. This demonstrates that it is possible to decouple G fusion activity and receptor recognition. We also show that although VSV can use alternative receptors of the LDL-R family, G mutants affected in their CR domain binding site cannot rescue a recombinant VSV lacking the G gene. Those data indicate that the only receptors of VSV are members of the LDL-R family and that G has specifically evolved to interact with their CR domains. This work provides structural insights on the interaction between G and host cell receptors and paves the way for the design of recombinant G with an altered tropism. 6 NSV 2018, Verona – Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 5 DELINEATING INTERACTION OF FILOVIRAL GP WITH ITS ENDOSOMAL RECEPTOR NPC1 BY IN SITU PROXIMITY LIGATION Eva Mittler* 1, Rohit K. Jangra1, Tanwee Alkutkar1, Kartik Chandran1 1Microbiology and Immunology, ALBERT EINSTEIN COLLEGE OF MEDICINE, New York, United States Abstract: Filoviruses are emerging zoonotic pathogens that cause outbreaks of lethal hemorrhagic fever in sub-Saharan Africa. The development of effective countermeasures against these agents is hindered by our limited understanding of filovirus-host molecular interactions required for viral entry and infection. For host cell entry, virions traffic to late endosomes/lysosomes (LE/LY), where the viral surface glycoprotein GP gains access to multiple essential host factors: GP is proteolytically processed by cathepsin B/L, and a cleaved form of GP (GPCL) binds to the critical intracellular receptor Niemann-Pick C1 (NPC1). Mechanistic studies of this indispensable binding step have been limited to a truncated, soluble form of a single domain in NPC1, domain C, as robust cell-based assays assessing interaction of GPCL with full-length NPC1 in its native context have been lacking. Here, we developed an in situ assay to monitor GPCL:NPC1 binding in infected cells. Subcellular visualization of this interaction at single molecule resolution was based on the principle of DNA-guided, antibody- + mediated in situ proximity ligation. GPCL:NPC1 interaction was restricted to the lumina of NPC1 LE/LY, and was blocked by disruption of GP’s proteolytic cleavage or GPCL:NPC1 interface formation. Testing the effect of FDA-approved small molecule inhibitors on proximity ligation revealed that drug treatments significantly disrupted virus entry and GPCL:NPC1 binding by distinct mechanisms. In summary, our in situ proximity ligation assay allows us to monitor GPCL:NPC1 engagement in intact