Sindbis virus, and enveloped RNA virus, is a benign member of a large group of medically important human and veterinary pathogens. Our studies are directed at understanding the molecular mechanisms involved in viral RNA replication, virus- induced modulation of host macromolecule synthesis, and virion assembly. We have constructed a cDNA clone of Sindbis virus which can be transcribed in vitro to produce infectious RNA molecules. This cloned will be used to map rigorously the lesions in mutants which have already been characterized and to create new mutants for further study. In particular, we will map the conditional mutations responsible for specific defects in RNA synthesis and virion assembly, and therefore assign these mutant phenotypes to specific viral nonstructural or structural peptides. Of the four Sindbis nonstructural proteins, nsP4 is of particular interest. This protein contains significant sequence homology with nonstructural proteins from all plant and animal (+) strand RNA viruses examined to date, and is therefore believed to play a central role in RNA replication. We will use both site-directed and saturation mutagenesis of the infectious clone to create a new catalog of silent, lethal, and conditional mutants in nsP4. Conditional mutants will be characterized to gain a better understanding of the function of nsP4 in RNA replication. We will also study methods to improve synthesis in vitro of functional RNA transcripts and to increase the efficiency with which they can be introduced into large numbers of cells. These improvements may allow the study of lethal mutations produced by in vitro mutagenesis of the infectious clone, and will also be of use to others studying genes by in vitro transcription and transfection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI024134-03
Application #
3136812
Study Section
Experimental Virology Study Section (EVR)
Project Start
1987-02-01
Project End
1990-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
3
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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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