Alphaviruses constitute a group of widely distributed, significant human and animal pathogens that differ in their ability to cause disease, but are believed to have similar life cycles, replication strategies and interactions with host cells. Sindbis virus (SIN), one of the least pathogenic among the alphaviruses, is to date the most valuable source of information about fundamental issues of alphavirus replication and ? pathogenesis on the molecular and cellular levels. Most results from studies of this virus are applicable to other members of the genus. In our recent research, we succeeded in designing a combination of methods that allow us to distinguish between the defects in plus- and minus-strand RNA synthesis during replication of SIN RNA. We found that the 5'UTR of the viral genome is an essential element of the promoter for negative-strand RNA synthesis that starts in the 3' end. Based on this finding we suggested a new model of initiation of SIN genome replication. We also generated a collection of mutants with a defective 5' cis-acting element in the genome RNA, which were unable to grow in mosquito cells, and then selected the pseudorevertants that resumed growth in the cells of insect origin, but retained all of the original mutations. Now we distinguish two functional elements in the 5' end of the SIN genome: the 5'UTR that is an element of core promoter, and the 51nt CSE that functions as a replicational enhancer in vertebrate cells and is critical for replication in insect cells. In the proposed study, we will dissect further the RNA elements in the promoter for negative-strand RNA synthesis and identify proteins required for initiation of Sindbis virus genome replication. We will analyze i) the RNA motifs on the 5' and 3' ends of the viral RNA, which form the core promoter, and ii) host cell and viral proteins that bind to the core promoter and the replicational enhancer to form the replicative complex. We will explore the segment of SIN genome, required for replication in mosquito cells, and identify viral protein(s) or another RNA element interacting with this fragment. We will use the results to design new attenuated strains of Venezuelan and eastern equine encephalitis viruses. These viruses will contain irreversible changes in their genomes that will reduce pathogenicity, but retain the antigenic structure of the wild-type ancestors. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
7R01AI050537-06
Application #
7767351
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Repik, Patricia M
Project Start
2003-03-01
Project End
2009-05-07
Budget Start
2009-01-15
Budget End
2009-05-07
Support Year
6
Fiscal Year
2007
Total Cost
$78,402
Indirect Cost
Name
University of Alabama Birmingham
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Frolova, Elena I; Gorchakov, Rodion; Pereboeva, Larisa et al. (2010) Functional Sindbis virus replicative complexes are formed at the plasma membrane. J Virol 84:11679-95
Atasheva, Svetlana; Krendelchtchikova, Valentina; Liopo, Anton et al. (2010) Interplay of acute and persistent infections caused by Venezuelan equine encephalitis virus encoding mutated capsid protein. J Virol 84:10004-15
Gorchakov, Rodion; Frolova, Elena; Sawicki, Stanley et al. (2008) A new role for ns polyprotein cleavage in Sindbis virus replication. J Virol 82:6218-31
Volkova, Eugenia; Frolova, Elena; Darwin, Justin R et al. (2008) IRES-dependent replication of Venezuelan equine encephalitis virus makes it highly attenuated and incapable of replicating in mosquito cells. Virology 377:160-9
Gorchakov, Rodion; Garmashova, Natalia; Frolova, Elena et al. (2008) Different types of nsP3-containing protein complexes in Sindbis virus-infected cells. J Virol 82:10088-101
Atasheva, Svetlana; Garmashova, Natalia; Frolov, Ilya et al. (2008) Venezuelan equine encephalitis virus capsid protein inhibits nuclear import in Mammalian but not in mosquito cells. J Virol 82:4028-41
Garmashova, Natalia; Atasheva, Svetlana; Kang, Wenli et al. (2007) Analysis of Venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription. J Virol 81:13552-65
Michel, Gilles; Petrakova, Olga; Atasheva, Svetlana et al. (2007) Adaptation of Venezuelan equine encephalitis virus lacking 51-nt conserved sequence element to replication in mammalian and mosquito cells. Virology 362:475-87
Garmashova, Natalia; Gorchakov, Rodion; Volkova, Eugenia et al. (2007) The Old World and New World alphaviruses use different virus-specific proteins for induction of transcriptional shutoff. J Virol 81:2472-84
Atasheva, Svetlana; Gorchakov, Rodion; English, Robert et al. (2007) Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J Virol 81:5046-57

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