Background:  It was recently proposed that certain HIV-1 RT mutations in the RNase H domain increase nucleoside reverse transcriptase inhibitor (NRTI) -resistance by reducing RNase H activity. The reduced RNA degradation could boost resistance by increasing the time available for excision of incorporated NRTI. We studied the effect on excision of zidovudine (AZT) -5’-monophosphate (AZT-MP) from terminated primers by changing assay parameters such as pH, Mg²⁺ and AZT-MP concentrations. These alterations are considered to occur in lymphocytes used for drug susceptibility assays or in human cells.

Methods:  RT mutants were constructed containing the following mutations:  M41L, D67N, K70R, L210W, T215Y (TAM-1), M41L, D67N, K70R, T215Y, K219Q (TAM-2) and M41L, T69S-SG, L210W, T215Y (69S-SG). ATP-mediated excision of AZT was studied using a non-radioactive RT assay with solid bound prA/odT template/primer and 5 mM ATP. Free Mg²⁺ concentration was reduced from RT assay optimal of 7 to 1 mM, pH was lowered from 7.5 to 7.0, and AZT-MP was included at 1 mM. These effects on RNase H activity were also measured using a radioactive solid bound prA/odT assay.

Results:  In the RT assay, fold resistance to AZT-TP was determined as the ratio of mutant to wild type IC₅₀. A 1.5- to 2-fold increase of ATP-dependent resistance to AZT-TP for TAM-1/2 and 69S-SG was noted by reducing free Mg²⁺ or adding AZT-MP. At pH 7.0, the increase was overall more evident, in particular with the 69S-SG mutant demonstrating a 3-fold increase (from an 8- to 24-fold IC₅₀ ratio). In the RNase H assay, a 20 to 40% decrease in activity for each of the above factors was observed. In comparison to RT activity, lower RNase H activity was found with pH 7.0 and AZT-MP, but similar decreases was noted at low Mg²⁺ concentrations.

Conclusions:  High AZT resistance in cell-based systems expressing TAM-containing HIV1-RT has previously been attributed to AZT’s bulky azido group preventing dead-end complex formation. It is likely that low intracellular pH, high intracellular levels of AZT-MP, or differences between physiological and enzymatic assay Mg²⁺ levels also are responsible for AZT resistance. These initial results suggest that differences between physiological and cell-based or enzymatic assay levels of these factors should be considered when measuring NRTI resistance, and support further studies to determine if these factors affect RNase H activity in promoting enhanced excision.