Abstract

: HIV-1 RT continues to be a key target for anti-HIV therapy. The long term goal of our project is to comprehensively define HIV RT function and the mechanism of drug resistance. There is an increasing realization that proper positioning of template-primer is central to several key aspects of RT function. We intend to delineate the functional role of three specific interactions between HIV-1 RT and the template-primer. These contact points lie along the template-primer cleft, in front of the active site (contacting the template 5'-overhang), behind the active site (contacting the template at penultimate basepair), or further away in the minor groove (contacting the primer at 3rd to 6th basepairs) respectively.
Three specific aims are listed. First, we will study the fingers subdomain-template contacts near the active site via nested deletions of 33-[M loop of HIV-1 RT created to disrupt the proposed interactions with the template overhang and/or the dNTP. In addition to testing these mutants for dNTP-binding, accuracy of dNTP insertion, processivity and strand transfer, we will also test the role of J33-j34 loop as a determinant of the natural high susceptibility of HIV-1 RT to 2',3'-dideoxyNTPs. The mutation rate of multi-dideoxynucleoside analog resistant RT variants and whether a proofreading-like activity contributes to the overall fidelity will be tested. Second, we will investigate the role of the template grip 135a, which forms a close contact with the penultimate basepair of the template-primer, in RT function. We will both attempt to delineate the mechanism of frameshift mutagenesis by HIV-1 RT, the precise role of template grip 135a in frameshift fidelity and the mechanistic basis for its global influence on fidelity. Altered template grip mutants of HIV-1 RT will be tested for effects on affinity to the template-primer duplex, processivity, RNase H activity and strand transfer. Lastly, we will test whether the thumb helix clamp involving aH and cxl helices indeed serves a critical function in enzyme translocation subsequent to each dNTP addition cycle. We will also test its role in processive polymerization and in frameshift fidelity.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030861-10
Application #
6724786
Study Section
Special Emphasis Panel (ZRG1-AARR-1 (01))
Program Officer
Ussery, Michael A
Project Start
1991-09-01
Project End
2006-03-30
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
10
Fiscal Year
2004
Total Cost
$496,596
Indirect Cost

  Institution

Name
Albert Einstein College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461

  Publications

Garforth, Scott J; Kim, Tae Woo; Parniak, Michael A et al. (2007) Site-directed mutagenesis in the fingers subdomain of HIV-1 reverse transcriptase reveals a specific role for the beta3-beta4 hairpin loop in dNTP selection. J Mol Biol 365:38-49
Drosopoulos, William C; Prasad, Vinayaka R (2007) The active site residue Valine 867 in human telomerase reverse transcriptase influences nucleotide incorporation and fidelity. Nucleic Acids Res 35:1155-68
Mandal, Dibyakanti; Dash, Chandravanu; Le Grice, Stuart F J et al. (2006) Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function. Nucleic Acids Res 34:2853-63
Drosopoulos, William C; Direnzo, Roberto; Prasad, Vinayaka R (2005) Human telomerase RNA template sequence is a determinant of telomere repeat extension rate. J Biol Chem 280:32801-10
Hehl, Eric A; Joshi, Pheroze; Kalpana, Ganjam V et al. (2004) Interaction between human immunodeficiency virus type 1 reverse transcriptase and integrase proteins. J Virol 78:5056-67
Rezende, Lisa F; Prasad, Vinayaka R (2004) Nucleoside-analog resistance mutations in HIV-1 reverse transcriptase and their influence on polymerase fidelity and viral mutation rates. Int J Biochem Cell Biol 36:1716-34
Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R (2003) Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template. J Virol 77:5837-45
Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R (2003) Substitutions at Phe61 in the beta3-beta4 hairpin of HIV-1 reverse transcriptase reveal a role for the Fingers subdomain in strand displacement DNA synthesis. J Mol Biol 325:443-59
Fisher, Timothy S; Joshi, Pheroze; Prasad, Vinayaka R (2002) Mutations that confer resistance to template-analog inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase lead to severe defects in HIV replication. J Virol 76:4068-72
Fisher, Timothy S; Prasad, Vinayaka R (2002) Substitutions of Phe61 located in the vicinity of template 5'-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase. J Biol Chem 277:22345-52

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