The long term objective of this proposal is to determine the mechanism by which the single-stranded RNA genome of poliovirus, the prototype of prototype of picornaviruses, is replicated in infected cells. The hypothesis that poliovirus genome-linked protein, VPg (or a precursor to VPg, Pre-VPg) is used as a primer for initiating synthesis of poliovirus plus- and minus-strand RNAs will be tested. The virus-specific proteins 3Dpol (RNA-dependent RNA polymerase) and Pre-VPg and a host a cell protein (host factor, HF) involved in viral replication will be purified using various chromatographic techniques. Purified proteins and appropriate serological reagents will be used to study the mechanism of viral RNA replication in vitro. Specifically, the requirements for uridylylation of Pre-VPg will be examined using biochemical and serological techniques. Whether uridylylated Pre-VPg (Pre-Vpg-pU or Pre-VPg-pUpU) can be used as a protein-nucleotide primer for in vitro synthesis of poliovirus plus- and minus-strand RNAs will be determined. The protein-protein interaction of 3Dpol,HF and Pre-VPg will be studied using biochemical and serological techniques. The mechanism of viral RNA replication will be further studied using mutant polioviruses defective in RNA synthesis. The normal function of host factor in uninfected cells will be examined. Since a double-stranded RNA (dsRNA)-activated protein kinase activity which phosphorylates the alpha-subunit of eukaryotic initiation factor-2 (eIF-2) copurifies with host factor, the exact relationship between host factor and dsRNA-activated kinase will be examined using biochemical and serological techniques. Furthermore, the mechanism by which poliovirus prevents increased phosphorylation of eIF02lalpha despite activation of dsRNA- activated kinase in infected cells, will be examined. 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 human and animals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI018272-09A1
Application #
3127789
Study Section
Experimental Virology Study Section (EVR)
Project Start
1981-09-30
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
9
Fiscal Year
1990
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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