[AEGiS-3HIVDRW: 34] BIOCHEMICAL ANALYSIS OF HIV-1 REVERSE TRANSCRIPTASE WITH MUTATIONS AT MET 184 IN THE HIGHLY CONSERVED YMDD REGION

3rd International Workshop on HIV Drug Resistance

2-5 August 1994, Kauai, Hawaii, USA

BIOCHEMICAL ANALYSIS OF HIV-1 REVERSE TRANSCRIPTASE WITH MUTATIONS AT MET 184 IN THE HIGHLY CONSERVED YMDD REGION

Int Wkshop HIV Drug Res 1994 Aug 2-5;3:35 (abstract no. 34)

JE Wilson^*, TB Caligan^*, JL Martin^*, A. Aulabaugh^*, LL Wright^*, S McPherson^**, JK Wakefield^**, S Jablonski^**, CD Morrow^**, and PA Funnan^*
^*Wellcome Research Laboratories, Research Triangle Park, NC, USA; ^**University of Alabama at Birmingham, Birmingham, AL, USA

A met to val mutation at position 184 in the highly conserved YMDD region of the human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) has been associated with the development of resistance to several nucleoside analogs including 3TC, FTC, ddI and ddC. This mutation was selected during in vitro passage in the presence of these drugs. The mutation at M184 to val was introduced in recombinant BH10 HIV-1 RT. The protein was expressed in E.coli BL21 and purified using phosphocellulose and DEAE cellulose chromatography. The relative purity of the enzyme was judged to be 95% by Coornassie Blue staining of SDS-polyacrylamide gels. Steady-state enzyme assays were performed with purified wt and the variant HIV-1 RT using natural dNTPs as well as chain-terminating 1-β-L and 1-β-D nucleotide analogues. Although the K_m and the k_cat values determined for natural substrates using the wt and the variant RT were similar, K_i values exhibited by the nucleotide analogs tested were dependent on absolute configuration. Using M184V RT, two- to five-fold elevated K_i values were obtained for 1-β-D analogues using a DNA primed RNA template, however, K_i values were not elevated when using a DNA primed RNA template. The K_i values exhibited by the corresponding 1-β-L configuration were elevated a 100- to 1000-fold under equivalent conditions. The origin of the extreme stereochemical dependence of substrate recognition and utilization by the variant enzyme is presently under study.

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1994-08-02
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