The applicant proposes to use an in vitro procedure that detects drug-resistant genetic variants of HIV reverse transcriptase (RT) expressed in bacteria. Employing a recently developed, novel in situ colony screening assay for HIV RT, substrate analog inhibitor-resistant HIV RT enzymes will be isolated. Cross-resistance of such mutant RTs to other drugs will be determined to help plan combination therapy. Selected mutations will be rebuilt into an infectious molecular clone of HIV and will be tested for replication competence. This will be done in an attempt to test the in vivo relevance of the mutant RTs with decreased sensitivity to substrate analog inhibitors obtained by in situ colony screening. Furthermore, the investigator proposes to characterize mutant RTs with respect to the site of mutation, rates of misincorporation and other aspects of DNA polymerization. To gain further insights into substrate-binding sites, residues in and around the mutations will be further examined by specific mutagenesis. Resistance detected biochemically may or may not reflect resistance during replication in vivo. To ascertain the ability to anticipate mutations that arise in vivo by utilizing this simple in vitro approach, viruses will be isolated from patients who are under therapy in AIDS clinical trials and their RT sequences will be tested for the presence of mutations that lead to resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030861-04
Application #
2065935
Study Section
AIDS and Related Research Study Section 3 (ARRC)
Project Start
1991-09-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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
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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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