Endogenous reverse transcriptase assays reveal synergy between combinations of the M184V and other drug resistance-conferring mutations in interactions with nucleoside analog triphosphates.

Resistance of HIV-1 reverse transcriptase (RT) to nucleoside analogs (e.g. AZT, ddC and 3TC) is conferred by various amino acid substitutions or combinations thereof on the RT molecule. The M184V mutation, that confers high and low-level resistance to 3TC and ddC, respectively, can restore sensitivity to AZT when introduced into RT against a background of AZT-resistance. The K65R mutation, that confers low level resistance to both 3TC and ddC, can also restore sensitivity to AZT. This information is of potential utility in choosing combinations of anti-viral drugs for clinical use. To explore this subject further, we have used an endogenous RT reaction to study mutated viruses containing M184V alone or M184V combined with each of the K65R, E89G or both the M41L and T215Y substitutions. Endogenous assays possess the advantage of utilizing genomic RNA as template in a reaction mixture that includes each of tRNALys.3 and viral nucleocapsid protein, necessary for specific initiation of reverse transcription, as well as all other viral proteins that might impact on this process. We now show that viruses containing both M184V and K65R displayed synergistic resistance to 3TC triphosphate (3TCTP), while the same combination yielded the same level of resistance to ddC triphosphate (ddCTP) as that manifested by K65R alone. The combination of M184V and E89G displayed synergistic resistance against ddCTP but not 3TCTP, while viruses containing only E89G were highly resistant to 3TCTP and displayed low-level resistance to ddCTP. The results show that endogenous RT assays can reveal variable synergistic, antagonistic, or neutral effects in regard to drug sensitivity, depending on the presence of specific amino acid substitutions in RT itself.