The goal of this investigation is to understand the mechanism of replication of picornaviruses typified by poliovirus. Proteins involved in viral RNA replication will be purified using various chromatographic techniques. Purified proteins as well as serological reagents will be used to determine the role of viral and host proteins in the initiation of poliovirus minus-stranded RNA synthesis. The hypothesis that poliovirus genome-linked protein, VPg (or its precursors) is used as a primer for initiating poliovirus plus- and minus-strand RNA synthesis will be tested using biochemcal techniques and an antibody to VPg. Roles of host factor (HF) and the virus-specific RNA polymerase (P63, replicase) in this reaction will be examined using biochemical and serological means. Specific agents, which selectively inhibit initiation of RNA synthesis as well as phosphorylation of a 63K protein in replicase preparation, will be used to study the involvement of protein phosphorylation in initiation of viral RNA replication. Biochemical and serological techniques will be used to determine if the kinase activity associated with viral polymerase is actually due to P63. Poliovirus ts-mutants that are defective in RNA synthesis will be used in complementation studies to furter define the roles of viral proteins in RNA replication. Molecular organization of the enzymatic machinery responsible for RNA replication will be studied by biochemical and immunochemical analysis of the isolated active replicase complex formed by mixing purified viral and host proteins in vitro. Also anti-VPg and anti-HF antibodies will be used to immunoprecipitate proteins complexed with HF and/or VPg from crude, soluble 7S replicase complex. Purified proteins will be used to study specific binding of polymerase to virion RNA using a recently developed RNA binding assay. Specificity of transcription of polio RNA (by the replicase) compared to other RNAs will be determined using biochemical and serological methods. The long-term goal of this research is to understand the mechanism of replication of picornaviruses. Studies will be continued to understand the mechanism of replication of poliovirus plus-strand RNA synthesis on minus-strand RNA template. Elucidation of the mechanism of replication of poliovirus will certainly fill the remaining large gap in knowledge concerning replication of this medically important virus group which includes those inducing common cold, infectious hepatitis, encephalomyocarditis and foot-to-mouth disease in humans and animals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI018272-06
Application #
3127791
Study Section
Experimental Virology Study Section (EVR)
Project Start
1981-09-30
Project End
1989-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
6
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Das, S; Ott, M; Yamane, A et al. (1998) Inhibition of internal entry site (IRES)-mediated translation by a small yeast RNA: a novel strategy to block hepatitis C virus protein synthesis. Front Biosci 3:D1241-52
Echeverri, A; Banerjee, R; Dasgupta, A (1998) Amino-terminal region of poliovirus 2C protein is sufficient for membrane binding. Virus Res 54:217-23
Das, S; Kenan, D J; Bocskai, D et al. (1996) Sequences within a small yeast RNA required for inhibition of internal initiation of translation: interaction with La and other cellular proteins influences its inhibitory activity. J Virol 70:1624-32
Echeverri, A C; Dasgupta, A (1995) Amino terminal regions of poliovirus 2C protein mediate membrane binding. Virology 208:540-53
Datta, U; Dasgupta, A (1994) Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro. J Virol 68:4468-77
Das, S; Coward, P; Dasgupta, A (1994) A small yeast RNA selectively inhibits internal initiation of translation programmed by poliovirus RNA: specific interaction with cellular proteins that bind to the viral 5'-untranslated region. J Virol 68:7200-11
Coward, P; Dasgupta, A (1992) Yeast cells are incapable of translating RNAs containing the poliovirus 5' untranslated region: evidence for a translational inhibitor. J Virol 66:286-95
Kliewer, S; Muchardt, C; Gaynor, R et al. (1990) Loss of a phosphorylated form of transcription factor CREB/ATF in poliovirus-infected cells. J Virol 64:4507-15
Clark, M E; Dasgupta, A (1990) A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells. Mol Cell Biol 10:5106-13
Kliewer, S; Garcia, J; Pearson, L et al. (1989) Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity. J Virol 63:4616-25

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