The goal of this project since its inception has been to understand virus structures, as a foundation for describing molecular events in virus assembly and entry. This renewal concerns entry. Advances in electron microscopy, in live-cell imaging, and in single-molecule detection allow us now to add to our structural "snapshots" the dimension of time and the context of a cell. Some properties of double-strand RNA (dsRNA) viruses offer particular advantages for studying non-enveloped virus entry;the flaviviruses offer related advantages for analyzing viral membrane fusion. Structures determined during the last grant period give us the information with which to ask precise questions about molecular mechanism. (1) Non- enveloped viral entry. We can follow rotavirus entry by live-cell fluorescence microscopy with particles bearing spectrally distinct labels on VP4 (the membrane perforation protein) and VP7 (the Ca2+ sensor for outer-layer uncoating). We can also use electron cryotomography (cryoET) to obtain medium-resolution images of entering particles. We will combine these approaches with information from the structure of the virion to work out the mechanisms of vesicular uptake, membrane perforation, and inner-particle release into the cytosol -- the sequence of events shown by our studies so far. (2) Enveloped-virus membrane fusion. We have developed a single-particle assay, to follow the time course of individual hemifusion and fusion events. We have shown how we can interpret kinetics of fusion, studied at the single-particle level, to probe the mechanism by which fusion proteins catalyze merger of two membrane bilayers. We will use this approach to study fusion of the non-infectious, recombinant virus-like particles that can be prepared with dengue and West Nile virus envelope proteins. The properties of these particles, in particular the ease of generating particles with a mutated envelope protein, will allow us to analyze steps in fusion that have not previously been accessible to single-particle kinetic analysis.
Rotaviruses and flaviviruses are both important human pathogens. Moreover, the rationale for the proposed work goes beyond implications for vaccines or anti-virals, as better understanding of viral entry will be valuable for design of gene therapy vectors and for delivery of targeted therapeutics (e.g., for cancer).
|Estrozi, Leandro F; Settembre, Ethan C; Goret, Gael et al. (2013) Location of the dsRNA-dependent polymerase, VP1, in rotavirus particles. J Mol Biol 425:124-32|
|Settembre, Ethan C; Chen, James Z; Dormitzer, Philip R et al. (2011) Atomic model of an infectious rotavirus particle. EMBO J 30:408-16|
|Aoki, Scott T; Trask, Shane D; Coulson, Barbara S et al. (2011) Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry. J Virol 85:10509-17|
|Kim, Irene S; Trask, Shane D; Babyonyshev, Marina et al. (2010) Effect of mutations in VP5 hydrophobic loops on rotavirus cell entry. J Virol 84:6200-7|
|Trask, Shane D; Kim, Irene S; Harrison, Stephen C et al. (2010) A rotavirus spike protein conformational intermediate binds lipid bilayers. J Virol 84:1764-70|
|Abraham, Jonathan; Corbett, Kevin D; Farzan, Michael et al. (2010) Structural basis for receptor recognition by New World hemorrhagic fever arenaviruses. Nat Struct Mol Biol 17:438-44|
|Wolf, Matthias; Garcea, Robert L; Grigorieff, Nikolaus et al. (2010) Subunit interactions in bovine papillomavirus. Proc Natl Acad Sci U S A 107:6298-303|
|Chen, James Z; Settembre, Ethan C; Aoki, Scott T et al. (2009) Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. Proc Natl Acad Sci U S A 106:10644-8|
|Aoki, Scott T; Settembre, Ethan C; Trask, Shane D et al. (2009) Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab. Science 324:1444-7|
|Lu, Xiaohui; McDonald, Sarah M; Tortorici, M Alejandra et al. (2008) Mechanism for coordinated RNA packaging and genome replication by rotavirus polymerase VP1. Structure 16:1678-88|
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