Abstract

The goal of this research is to define biochemical mechanisms of avian retrovirus replication through the study of the interactions of viral proteins with nucleic acids. These multidisciplinary model studies have direct application to development of anti-HIV agents.
The first aim of this proposal is to study the interaction of the viral gag nucleocapsid (NC) and matrix (MA) proteins and their Pr76gag precursor with viral RNA and to evaluate their role in virion assembly. Molecular genetic techniques will be used to make amino acid substitutions at highly conserved or biochemically important residues of the NC and MA proteins. The effect these changes have on viral replication in vivo and on the biochemical properties of the mutant proteins purified either from virus or bacteria will be determined. The role of the Pr76gag or NC and MA proteins in the recognition of specific packaging signals in viral RNA will also be studied.
The second aim of this proposal is to elucidate the mechanism of action of the DNA endonuclease associated with the viral integration (IN) protein and its role in viral DNA integration. Biochemical and molecular genetic experiments are outlined to relate the sequences at the termini of long terminal repeats (LTR) of viral DNA required for specific cleavage in vitro by the IN protein to those required for integration in vivo. This will be accomplished by analyzing the effect of small U5 LTR deletions on both cleavage and on integration of viral DNA. The requirement for inverted repeat sequences in the LTR in linear and supercoiled DNA substrates for IN protein activity and integration in vivo will be evaluated. Reconstitution of a viral DNA integration system in vitro using purified components will be attempted. In a related project, the role of secondary structure in the U5 region of viral RNA for initiation of reverse transcription will be studied. The observation that such structures potentially exist in other retrovirus RNAs including HIV suggest that this is a general phenomenon of replication.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA038046-07
Application #
3176063
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1984-07-01
Project End
1994-05-31
Budget Start
1990-06-01
Budget End
1991-05-31
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost

  Institution

Name
Case Western Reserve University
Department
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106

  Publications

Johnson, Michael; Morris, Shannon; Chen, Aiping et al. (2004) Selection of functional mutations in the U5-IR stem and loop regions of the Rous sarcoma virus genome. BMC Biol 2:8
Brin, Elena; Leis, Jonathan (2002) HIV-1 integrase interaction with U3 and U5 terminal sequences in vitro defined using substrates with random sequences. J Biol Chem 277:18357-64
Brin, Elena; Leis, Jonathan (2002) Changes in the mechanism of DNA integration in vitro induced by base substitutions in the HIV-1 U5 and U3 terminal sequences. J Biol Chem 277:10938-48
Morris, Shannon; Johnson, Michael; Stavnezer, Ed et al. (2002) Replication of avian sarcoma virus in vivo requires an interaction between the viral RNA and the TpsiC loop of the tRNA(Trp) primer. J Virol 76:7571-7
VerPlank, L; Bouamr, F; LaGrassa, T J et al. (2001) Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci U S A 98:7724-9
Xiang, Y; Thorick, R; Vana, M L et al. (2001) Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity. J Virol 75:6016-21
Hindmarsh, P; Johnson, M; Reeves, R et al. (2001) Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro. J Virol 75:1132-41
Brin, E; Yi, J; Skalka, A M et al. (2000) Modeling the late steps in HIV-1 retroviral integrase-catalyzed DNA integration. J Biol Chem 275:39287-95
Hindmarsh, P; Ridky, T; Reeves, R et al. (1999) HMG protein family members stimulate human immunodeficiency virus type 1 and avian sarcoma virus concerted DNA integration in vitro. J Virol 73:2994-3003
Morris, S; Leis, J (1999) Changes in Rous sarcoma virus RNA secondary structure near the primer binding site upon tRNATrp primer annealing. J Virol 73:6307-18

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