Retroviral reverse transcriptases contain a DNA polymerase activity and an RNase H activity that cooperate to copy the viral single-stranded RNA genome, producing a linear double-stranded DNA that is subsequently integrated into the genome of the host cell. The long term objective of this program is to understand in molecular detail all of the steps in the process of reverse transcription by both Moloney murine leukemia virus (M- MuLV) and human immunodeficiency virus type 1 (HIV-1). A combined biochemical and genetic approach is proposed to investigate (i) the multiple roles of RNase H in reverse transcription, and (ii) the capability of reverse transcription, and (ii) the capability of reverse transcriptase to displace the nontemplate strand during DNA synthesis on a nicked DNA template. Studies to elucidate the basis for the specificity of plus- strand priming will involve both a search for reverse transcriptase mutants that retain non-specific RNase H activity but are impaired for primer generation, and a characterization of the in vivo phenotype of mutations in the polypurine tract. The specificity of RNase H for removal of both the tRNA primer for minus strands and the polypurine tract primer for plus strands will be investigated in relation to the terminal structures required for integration. The strand displacement capability of the polymerase activity of HIV-1 reverse transcriptase will be determined and compared with recent results from the M-MuLV system. The ability of the polymerases to displace an RNA strand and the effects of the nucleocapsid protei on displacement synthesis will be investigated. The kinetics, processivity, and pausing patterns during displacement synthesis along with biochemical studies on the nature of the interaction between the polymerase and the nontemplate strand will provide data to distinguish a passive from an active mechanism for the displacement reaction. Experiments will be undertaken to isolate and characterize mutants that are defective in helix unwinding as a means to identify the domains of the reverse transcriptases responsible for displacement synthesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA051605-09
Application #
2608065
Study Section
AIDS and Related Research Study Section 3 (ARRC)
Program Officer
Cremer, Kenneth J
Project Start
1989-09-30
Project End
1999-11-30
Budget Start
1998-01-01
Budget End
1998-11-30
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2010) Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H. J Mol Biol 397:161-78
Champoux, James J; Schultz, Sharon J (2009) Ribonuclease H: properties, substrate specificity and roles in retroviral reverse transcription. FEBS J 276:1506-16
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2009) Preferred sequences within a defined cleavage window specify DNA 3' end-directed cleavages by retroviral RNases H. J Biol Chem 284:32225-38
Schultz, Sharon J; Zhang, Miaohua; Champoux, James J (2004) Recognition of internal cleavage sites by retroviral RNases H. J Mol Biol 344:635-52
Winshell, Jamie; Paulson, Benjamin A; Buelow, Ben D et al. (2004) Requirements for DNA unpairing during displacement synthesis by HIV-1 reverse transcriptase. J Biol Chem 279:52924-33
Lanciault, Christian; Champoux, James J (2004) Single unpaired nucleotides facilitate HIV-1 reverse transcriptase displacement synthesis through duplex RNA. J Biol Chem 279:32252-61
Winshell, J; Champoux, J J (2001) Structural alterations in the DNA ahead of the primer terminus during displacement synthesis by reverse transcriptases. J Mol Biol 306:931-43
Kelleher, C D; Champoux, J J (2000) RNA degradation and primer selection by Moloney murine leukemia virus reverse transcriptase contribute to the accuracy of plus strand initiation. J Biol Chem 275:13061-70
Schultz, S J; Zhang, M; Kelleher, C D et al. (2000) Analysis of plus-strand primer selection, removal, and reutilization by retroviral reverse transcriptases. J Biol Chem 275:32299-309
Schultz, S J; Zhang, M; Kelleher, C D et al. (1999) Polypurine tract primer generation and utilization by Moloney murine leukemia virus reverse transcriptase. J Biol Chem 274:34547-55

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