4th International Workshop on HIV Drug Resistance & Treatment Strategies Sitges, Spain, 12–16 June 2000

EFFECTS OF M41L AND T215Y MUTATIONS IN HIV-1 REVERSE TRANSCRIPTASE ON REMOVAL OF CHAIN-TERMINATORS FROM BLOCKED PRIMER/TEMPLATES

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):14 (Abstract 15

PR Meyer¹, I Pfeifer¹, S Matsuura¹, H Bazmi², AG So¹, JW Mellors² and WA Scott^1
1Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Fla.; and ²Department of Medicine, University of Pittsburgh, Pittsburgh, Pa., USA

OBJECTIVE: We have previously shown that HIV-1 reverse transcriptase (RT) containing the zidovudineresistance mutations D67N, K70R, T215F and K219Q has an increased ability to remove 3′-azido-3′deoxythymidine monophosphate (zidovudine-MP), and other chain-terminators, such as 2′,3′dideoxyadenosine monophosphate (ddAMP), from blocked primer/templates. This arises through elevated levels of nucleotide-dependent transfer of the chain-terminator to the nucleotide substrate, generating an extendible primer and a dinucleoside polyphosphate. We have now investigated the effects of the zidovudine-associated RT mutations M41L and T215Y on ATP- and pyrophosphate (PPi)-dependent unblocking of primer/templates terminated with zidovudine-MP or ddA-MP.

METHODS: Removal of zidovudine-MP was assessed by incubating 5′ [³²P]-labelled, zidovudine-MP-terminated primer/template with wild-type (WT) or mutant HIV-1 RT and either ATP or PPi, followed by primer/template extension with Klenow polymerase. Products were separated by electrophoresis and quantitated. Removal of ddA-MP was measured by incubating 3′ [³²P]-labelled, ddA-MP-terminated primer/template with WT or mutant RT and either ATP or PPi. The products of the reaction were separated by electrophoresis and Ap4ddA or ddA-TP quantitated. The relative catalytic efficiency for ATP-dependent removal (mutant k_cat/K_m,ATP divided by WT k_cat/K_m,ATP) was calculated for each mutant RT.

RESULTS: The relative catalytic efficiencies of ATP- dependent removal of zidovudine-MP by the mutant RTs were as follows: 41L RT, 1.3; 215Y, 1.8; 41L/215Y, 2.9; 67N/70R/215F/219Q, 4.0; and 67N/70R/215Y/219Q, 9.8. The ability of the mutant RTs to remove ddAMP from blocked primers through formation of Ap4ddA was also increased compared to WT. The relative catalytic efficiencies of ATP-dependent removal of ddA-MP were as follows: 41L RT, 1.6; 215Y, 4.4; 41L/215Y, 10; 67N/70R/215F/219Q, 9.4; and 67N/70R/215Y/219Q, 10. In contrast, there was no increase in PPi-dependent removal of either zidovudine- MP or ddA-MP by any mutant RT compared to WT.

CONCLUSIONS: The M41L mutation had little effect on ATP-dependent removal of chain-terminators by itself, but enhanced the increased removal conferred by the T215Y mutation. The M41L/T215Y mutations conferred an increase in ATP-dependent removal of zidovudine-MP to almost the same extent as the D67N/K70R/T215F/K219Q mutations. These results agree well with the relative effects that these mutations confer on HIV-1 phenotypic zidovudine resistance. Our data also suggests that the T215Y mutation, in the context of the D67N, K70R and K219Q mutations, confers a higher increase in ATP-dependent removal of zidovudine-MP than the T215F mutation. Taken together, our results indicate that increased ATP- dependent removal of zidovudine-MP from blocked DNA chains is an important mechanism of zidovudine resistance.

Download abstract

2000-06-12
15

Copyright © 2000 - International Medical Press Ltd. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the International Medical Press Ltd. 2-4 Idol Lane, London EC3R 5DD UK.