Crystallographic, biophysical, and molecular biological studies were performed by our group and collaborators on insect nodaviruses and picorna-like viruses. A number of observations were made that apparently relate to specific functions of the particle in the virus life cycle. This renewal application describes experiments in the area of cell biology as well as crystallography, biophysics and molecular virology, as efforts are made to define viral phenotypes on the basis of chemistry discerned from atomic resolution and in vitro studies. The essence of the proposal is to span the range from atoms to cells for simple, yet elegant, organisms, the non enveloped, ssRNA viruses of insects and plants. The variations observed in structure and hypothesized function lead naturally to an investigation of the evolution of these particles. Specifically we seek to: (1) Employ fluorescence and electron microscopy to determine the mode of entry of flock house virus (FHV) and, to employ biochemistry and biophysics to determine the role of the receptor in RNA release. (2) Employ in vivo expression of GFP fusion proteins in mammalian and drosophila cells to establish the role pf a membrane active peptide derived from the capsid protein of FHV by a maturation cleavage. (3) Determine the structure of the RNA directed RNA polymerase of FHV and establish its novel features that allow it to replicate and CAP RNA molecules in insect, plant, yeast and mammalian cells. (4) Investigate, by crystallography, cryoEM, baculovirus expression and site directed mutagenesis, factors that affect assembly, maturation and genome organization of insect and fish nodaviruses. (5) Determine the sites of FHV RNA replication, capsid protein synthesis and particle assembly by fluorescence and electron microscopy. (6) Establish evolutionary relationships among insect and fish nodaviruses and among insect and plant picorna and picorna-like viruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM034220-21
Application #
6730023
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Chin, Jean
Project Start
1984-12-01
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
21
Fiscal Year
2004
Total Cost
$421,330
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Routh, Andrew; Johnson, John E (2014) Discovery of functional genomic motifs in viruses with ViReMa-a Virus Recombination Mapper-for analysis of next-generation sequencing data. Nucleic Acids Res 42:e11
Johnson, John E (2013) Confessions of an icosahedral virus crystallographer. Microscopy (Oxf) 62:69-79
Veesler, David; Johnson, John E (2012) Virus maturation. Annu Rev Biophys 41:473-96
Routh, Andrew; Ordoukhanian, Phillip; Johnson, John E (2012) Nucleotide-resolution profiling of RNA recombination in the encapsidated genome of a eukaryotic RNA virus by next-generation sequencing. J Mol Biol 424:257-69
Routh, Andrew; Domitrovic, Tatiana; Johnson, John E (2012) Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus. Proc Natl Acad Sci U S A 109:1907-12
Baudoux, A-C; Hendrix, R W; Lander, G C et al. (2012) Genomic and functional analysis of Vibrio phage SIO-2 reveals novel insights into ecology and evolution of marine siphoviruses. Environ Microbiol 14:2071-86
Speir, Jeffrey A; Johnson, John E (2012) Nucleic acid packaging in viruses. Curr Opin Struct Biol 22:65-71
Banerjee, Manidipa; Speir, Jeffrey A; Kwan, Maggie H et al. (2010) Structure and function of a genetically engineered mimic of a nonenveloped virus entry intermediate. J Virol 84:4737-46
Odegard, Amy; Banerjee, Manidipa; Johnson, John E (2010) Flock house virus: a model system for understanding non-enveloped virus entry and membrane penetration. Curr Top Microbiol Immunol 343:1-22
Odegard, Amy L; Kwan, Maggie H; Walukiewicz, Hanna E et al. (2009) Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry. J Virol 83:8628-37

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