Elevated nucleotide-dependent primer unblocking by zidovudine-resistant HIV-1 reverse transcriptase leads to increased removal of zidovudine-MP from blocked primers
Originally Published on September 2, 1999
PR Meyer1, S Matsuura1, M Mian1, AG So1,2 and WA Scott1
ABSTRACT: Purpose: The mechanism of zidovudine resistance is still unclear. We have recently reported a novel activity of HIV-1 reverse transcriptase (RT) that can unblock chain-terminated primers through a nucleotide-dependent reaction. This occurs through transfer of the primer terminus to a nucleotide acceptor, such as ATP, through a pyrophosphorolysis-related reaction producing a shortened, extendible primer terminus and dinucleoside polyphosphate. We have studied the ability of wild-type RT and RT containing the zidovudine resistance mutations D67N/K70R/T215F/K219Q (mutant RT) to remove zidovudine-MP from blocked primers. We have also assessed the ability of foscarnet, a pyrophosphate analogue, to inhibit this reaction.
Methods: Primer extension was performed in the presence of zidovudine-TP, in the absence or presence of ATP. Production of dinucleoside polyphosphates or extendible 'rescued' primer was measured after incubation of zidovudine-MP-terminated primer/template, RT and ATP or GTP.
Results: In the absence of ATP, wild-type and mutant RT showed no difference in the primer extension pattern, indicating little difference in ability to discriminate against zidovudine-TP. However, in the presence of ATP, mutant RT was able to extend 1215% of primers past multiple termination sites compared to 0.20.5% for wild-type RT. This suggested that mutant RT had increased ability to remove zidovudine-MP through dinucleoside polyphosphate synthesis, producing an extendible primer and Ap4-zidovudine. Indeed, mutant RT had about a fourfold increased rate of both Ap4-zidovudine and Gp4-zidovudine synthesis and ATP- or GTP-induced rescue compared to wild-type RT. Foscarnet blocked dinucleoside polyphosphate synthesis and completely suppressed the zidovudine-resistant phenotype observed in the primer extension assay when present at a concentration of 12µM.
Conclusions: Our results indicate that removal of zidovudine-MP through nucleotide-dependent primer unblocking is part of the mechanism of zidovudine resistance. The concomitant use of foscarnet and zidovudine might be beneficial by suppressing zidovudine resistance and possible by potentiating the effect of zidovudine.
Supported by NIH AI31848, DK26206.
