2130-Pos Board B149 Conformational Dynamics of Single HIV Env Molecules on the Surface of Native Virions James Munro 1 , Jiang Zhu 2 , Jonathan Stuckey 2 , Peter Kwong 2 , Scott Blanchard 3 , Walther Mothes 1 . 1 Yale University, New Haven, CT, USA, 2 Vaccine Research Center, National Institutes of Allergy and Infectious Diseases, Bethesda, MD, USA, 3 Weill Cornell Medical College of Cornell University, New York, NY, USA. Entry of HIV is accomplished by the trimeric envelope spike (Env), a conforma- tional machine that avoids recognition by antibodies through a 2-receptor mechanism of activation, involving CD4 and a coreceptor. We introduced flu- orophores into distinct domains of gp120, the receptor-binding subunit of the spike, and monitored the conformational changes though single-molecule fluo- rescence resonance energy transfer (smFRET) measurements on the surface of infectious HIV virions. smFRET trajectories revealed transitions between three conformations intrinsically accessible to the unliganded Env. Binding of CD4 stabilized one of the pre-existing states. Binding of CD4 and the coreceptor- surrogate antibody 17b stabilized a second pre-existing state. Markov modeling of the smFRET trajectories indicated a preferred order to the conformational changes with the CD4-stabilized state an intermediate critical to achieving the likely coreceptor-stabilized state. Distances estimated from the observed FRET states were integrated with known structures of HIV Env to produce a temporally accurate simulation of gp120, as it activates the underlying gp41-fusion machinery. Our work provides a framework for understanding the stepwise activation of HIV Env through successive binding of the receptor and coreceptor. 2131-Pos Board B150 Influenza Virus Binds its Host Cell using Multiple Dynamic Interactions Revealed by Single Virus Force Spectroscopy and Force Probe Molecular Dynamics Andreas Herrmann 1 , Christian Sieben 1 , Christian Kappel 2 , Rong Zhu 3 , Christian Rankl 4 , Anna Wozniak 5 , Peter Hinterdorfer 3 , Helmut Grubmu ¨ller 2 . 1 Humboldt-University Berlin, Berlin, Germany, 2 Max Planck Institute for Biophysical Chemistry, Go ¨ttingen, Germany, 3 Johannes Kepler University, Linz, Austria, 4 Agilent Technologies-Austria, Linz, Austria, 5 JPK Instruments, Berlin, Germany. Influenza virus belongs to a wide range of enveloped viruses. Virus-host cell binding marks the first critical step of infection. Hence, forces involved in this process are essential. The major spike protein hemagglutinin (HA) binds sialic acid (SA) residues of glycoproteins and -lipids of the cell surface of host cells with dissociation constants in the millimolar range, indicating a multivalent binding mode. Here we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy based single molecule force spectroscopy re- vealed very low interaction forces in the order of ~10-25 pN. Further, the ob- servation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Force probe molecular dy- namics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor allowing to rational- ize influenza virus-cell binding quantitatively at molecular level. Simulations show that the receptor-binding domain of HA is very rigid and does not show any deformations during unbinding of the ligand, which does not argue for any cooperativity between HA monomers in binding. The observed unbinding pathways are diverse and contain zipper-like as well as all-or-none unbinding events. Notably, we did not find a clear preference for binding of the probed influenza strains to one of the cell lines differing in the structure of the SA receptors. Sieben et al. (2012) Proc Natl Acad Sci USA. 09:13626-13631 2132-Pos Board B151 Dengue Virus Capsid Protein Binding to Lipid Droplets and its Inhibition. towards a New Drug Target Filomena A. Carvalho 1 , Ivo C. Martins 1 , Fabiana A. Carneiro 2 , Iranaia Assunc ¸a ˜o-Miranda 2 , Andre ´ F. Faustino 1 , Miguel Castanho 1 , Ronaldo Mohana-Borges 2 , Fabio C.L. Almeida 2 , Andrea T. Da Poian 2 , Nuno C. Santos 1 . 1 Instituto de Medicina Molecular, Faculdade Medicina Universidade Lisboa, Lisbon, Portugal, 2 Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths yearly. No effective treatment is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essen- tial for viral replication. Atomic force microscopy (AFM)-based force spectros- copy measurements were performed with DENV C-functionalized AFM tips. DENV C-LDs interaction was found to be strong and dependent on the high in- tracellular potassium concentration. Inhibition of Na þ /K þ -ATPase in DENV- infected cells resulted in the dissociation of DENV C from LDs and a 50- fold inhibition of infectious virus production (but not of RNA replication), demonstrating the biological relevance of the K þ -dependence. Limited LDs proteolysis impaired DENV C-LDs interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the ma- jor DENV C ligand on the LDs surface. A peptide based on a conserved seg- ment of DENV C intrinsically disordered N-terminal domain (pep14-23) was shown by force spectroscopy to successfully inhibit DENV C-LDs interaction. These advances pave the way to drug development approaches, in which inhib- itory efficiency may be improved through lead optimization. A similar strategy may be used for other flaviviruses. Carvalho et al. (2012) J. Virol., 86, 2096-2108. Martins et al. (2012) Biochem. J., 444, 405-415. 2133-Pos Board B152 A Polymerase Super Complex within Vesicular Stomatitis Virus Jeffery A. Hodges 1 , Xiaolin Tang 1 , Michael Landesman 1 , John B. Ruedas 2 , Anil Ghimire 1 , Jacques Perrault 2 , Manasa V. Gudheti 1 , Jordan Gerton 1 , Saveez Saffarian 1 . 1 University of Utah, Salt Lake CIty, UT, USA, 2 San Diego State University, San Diego, CA, USA. The material properties of biological samples such as viruses are important, and properties such as elasticity can be determined with technologies like Atomic Force Microscopy (AFM). These material studies have a great impact on our understanding of viral assembly, budding and maturation regarding highly ordered viruses such as Vesicular Stomatitis Virus (VSV). VSV is a negative sense RNA virus which is studied widely as a model system for non-segmented negative sense RNA viruses. These viruses require the assem- bly and inclusion of multiple copies of their RNA-specific polymerase for transcription and replication in future generations. Despite their importance, little is known about their locality in a fully formed virion. While studying the elastic material property of intact wild-type VSV utilizing AFM; in con- junction with super resolution microscopy techniques; we have discovered that 20-70 polymerases stack at the blunt end of each virion near the 3’ tran- scriptional promoter and form a super complex with the polymerase cofactor proteins of the virus. Since these observations show that the super complex is located near the only transcriptional promoter of the mature virus, they imply a mechanism other than stochastic collision for the association of polymerase with the promoter for initial transcription of all the polymerase within the virus. 2134-Pos Board B153 Single Influenza Virus Tracking Reveals Cell Specific Modes of Movement - a Key to Understand Cellular Tropism Christian Sieben 1 , Nathanael Hoze 2 , Katharina Horst 1 , Christopher Wolff 1 , Maik J. Lehmann 1 , David Holcman 2 , Andreas Herrmann 1 . 1 Humboldt-University Berlin, Berlin, Germany, 2 Ecole Normale Supe ´rieure, Paris, France. Following single viruses on their journey through a host cell provides valuable insights that help to understand the infection process. Fluorescent labeling tech- niques together with high-resolution microscopic imaging allows tracking of single viruses on living cells with high temporal and spatial accuracy. Influenza A viruses were fluorescently labeled without interfering with their bi- ological activity. Single-virus trajectories were recorded on the surface and in- side living cells of different permissive cell lines. By using conventional computational methods we extracted the position of viruses within each image frame. In addition, we have applied a recently developed mathematical tool to further analyze a special type of recurrent trajectory obtained at the surface of living cells. using this approach we can identify and characterize microdomains on the cell surface. We show that the viruses are surprisingly mobile and dis- play a variety of different types of movement, which depend on the studied cell line. The cell surface was characterized by scanning electron microscopy (SEM) and revealed a unique topography that also strongly depends on the cell type and the cells growth state. By comparing trajectories obtained from two permissive cell lines we were able to correlate the distinctive behavior of single viruses with the cell surface ultra- structure. We used cells that are infected to a different degree and combine this Tuesday, February 5, 2013 415a