The research interests of my laboratory are centered on the molecular details of animal RNA virus replication and the interactions of these viruses with their vertebrate and invertebrate hosts. The experiments detailed in this proposal concern our current studies on Sindbis virus (SIN), type virus of the family Togaviridae. Several members of this family are agents of chronic arthropod-transmitted world health problems, and our long range goals include a thorough understanding of viral RNA replication and gene expression, virion assembly, and viral pathogenesis and immunity. We eventually hope to apply this information for design for antiviral therapies and effective vaccines. our previous in vivo studies on the assembly of functional RNA replication complexes have led to the development of an in vitro RNA replication system which is dependent upon addition of exogenous SIN plus-strand RNA. This system will be used to define RNA promoter elements and RNA-protein interactions critical for initiation of minus-strand synthesis, to test a current model for the regulation of SIN RNA replication and transcription, and to provide an assay for purification and characterization of the viral RNA-dependent RNA polymerase.
A second aim i s to elucidate and study the viral functions responsible for vertebrate host cell death. Preliminary work has demonstrated that stable mammalian cell lines can be isolated after transformation with SIN replicons expressing a dominant selectable marker. Genetic analysis of these adapted replicons should provide clues as to the viral elements responsible for shut off of host translation and transcription and induction of apoptosis. Relevant viral components will be used to identify interacting host components in an attempt to obtain a mechanistic understanding of how these processes occur. Incorporation of such adaptive mutations into SIN expression vectors should greatly extend their utility by minimizing perturbation of host-cell biology. A third major focus is to study the mechanisms by which two novel SIN isolates invade the CNS of weanling mice. Infectious clones have been constructed for closely related noninvasive/invasive pairs and the genetic loci responsible for neuroinvasion have been determined. For one neuroinvasive strain, peripheral infection appears to induce the production of a soluble mediator of blood-brain barrier breakdown. Elucidation of the mechanisms leading to mediator production as well as its characterization, possible purification,a nd identification could have potential applications for drug delivery to the CNS or, if this is a general mechanism used by other pathogens for CNS invasion, methods for neutralization of the mediator may be useful for preventing spread to the CNS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
7R37AI024134-14
Application #
6452992
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Meegan, James M
Project Start
1987-02-01
Project End
2006-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
14
Fiscal Year
2001
Total Cost
$408,562
Indirect Cost
Name
Rockefeller University
Department
Microbiology/Immun/Virology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Cristea, Ileana M; Carroll, John-William N; Rout, Michael P et al. (2006) Tracking and elucidating alphavirus-host protein interactions. J Biol Chem 281:30269-78
Gorchakov, Rodion; Frolova, Elena; Williams, Bryan R G et al. (2004) PKR-dependent and -independent mechanisms are involved in translational shutoff during Sindbis virus infection. J Virol 78:8455-67
Gorchakov, Rodion; Hardy, Richard; Rice, Charles M et al. (2004) Selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication. J Virol 78:61-75
De Francesco, Raffaele; Rice, Charles M (2003) New therapies on the horizon for hepatitis C: are we close? Clin Liver Dis 7:211-42, xi
Hardy, Richard W; Marcotrigiano, Joseph; Blight, Keril J et al. (2003) Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. J Virol 77:2029-37
Frolova, Elena I; Fayzulin, Rafik Z; Cook, Susan H et al. (2002) Roles of nonstructural protein nsP2 and Alpha/Beta interferons in determining the outcome of Sindbis virus infection. J Virol 76:11254-64
Kummerer, Beate M; Rice, Charles M (2002) Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 76:4773-84
Frolov, I; Hardy, R; Rice, C M (2001) Cis-acting RNA elements at the 5' end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis. RNA 7:1638-51
Qu, L; McMullan, L K; Rice, C M (2001) Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity. J Virol 75:10651-62
Frolov, I; Agapov, E; Hoffman Jr, T A et al. (1999) Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells. J Virol 73:3854-65

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