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

EFFECTS OF FOSCARNET RESISTANCE MUTATIONS IN HIV-1 REVERSE TRANSCRIPTASE: SUPPRESSION OF ZIDOVUDINE RESISTANCE THROUGH REDUCED REMOVAL OF ZIDOVUDINE-MP FROM BLOCKED PRIMER/TEMPLATES

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):13 (Abstract 14

PR Meyer¹, R Chopra¹, E Pendarvis¹, S Matsuura¹, H Bazmi², AG So¹, JW Mellors² and WA Scott^1
1Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, USA; and ²Department of Medicine, University of Pittsburgh, USA

OBJECTIVE: HIV-1 resistance to zidovudine is associated with increased ATP-dependent removal of zidovudine-MP from blocked primer/templates by mutant HIV-1 reverse transcriptase (RT) containing zidovudine resistance mutations. Some mutations in HIV-1 RT that are selected in response to other RT inhibitors have been shown to resensitize HIV-1 to zidovudine. Such mutations include W88G, E89K, Q161L and H208Y, which confer resistance to phosphonoformate (foscarnet), a pyrophosphate (PPi) analogue. We have studied the effects of these mutations, by themselves or in zidovudine-resistant backgrounds, on the ability of HIV-1 RT to remove chain-terminators from blocked primer/templates, either through ATP-dependent removal or through pyrophosphorolysis.

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 extension of unblocked primer/template by addition of exonuclease-free Klenow fragment of E. coli DNA polymerase I. Removal of ddAMP was measured by incubating 3′-[³²P]-labelled, ddA-MP-terminated primer/template with WT or mutant RT and either ATP or PPi, followed by quantitation of [³²P]-labelled Ap4ddA or ddA-TP, respectively. ATP-dependent removal by the mutant RTs were compared to that of WT RT by determining the relative catalytic efficiency (k_cat/K_m,ATP for mutant RT divided by k_cat/K _m,ATP for WT RT) for each mutant RT.

RESULTS: ATP-dependent removal of zidovudine-MP was decreased in RT containing foscarnet resistance mutations. The 88G mutation decreased ATP-dependent removal of zidovudine-MP from blocked primer/templates by RT containing the 41L/215Y or 67N/70R/215F/219Q mutations to levels similar to WT RT (relative catalytic efficiencies: 88G RT, 0.15; 67N/70R/215F/219Q RT, 4.0; 67N/70R/88G/215F/ 219Q RT, 0.98; 41L/215Y RT, 2.9; 41L/88G/215Y RT, 0.3). The 89K mutation decreased removal by about three- to four-fold (relative catalytic efficiencies: 67N/70R/215Y/219Q RT, 9.8; 67N/70R/89K/215Y/ 219Q RT, 2.9). The 161L and/or 208Y mutations decreased ATP-dependent removal by RT containing the 67N/70R/215F/219Q mutations by about two- to three-fold (relative catalytic efficiencies: 161L RT, 0.34; 67N/70R/215Y/219Q RT, 9.8; 67N/70R/161L/215Y/ 219Q RT, 4.1; 161L/208Y RT, 0.41; 67N/70R/161L/ 208Y/215Y/219Q RT, 4.9). There was a parallel decrease in ATP-dependent removal of ddA-MP conferred by the foscarnet resistance mutations. PPi-dependent removal of zidovudine-MP or ddA-MP was also decreased in enzymes containing foscarnet resistance mutations; however, in contrast to ATP-dependent removal, there was no increase in PPidependent removal reaction for any of the zidovudine-resistant RTs.

CONCLUSIONS: The decrease in ATP-dependent removal of zidovudine-MP from blocked primer/ templates conferred by foscarnet resistance mutations provides a likely explanation for the resensitization of zidovudine-resistant HIV-1 to zidovudine. The association between foscarnet resistance and decreased ATP-dependent and PPi-dependent primer rescue suggests that foscarnet, ATP and PPi may share a common binding site on the enzyme.

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2000-06-12
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