The Enzymatic Basis for Thymidine Analogue Resistance in HIV-1

Mutations at six sites in HIV-1 reverse transcriptase (RT) that have long been associated with resistance to zidovudine (AZT) are now referred to as thymidine analogue mutations (TAMs) because they help the virus escape from stavudine (d4T) as well. These mutations include M41L, D67N, K70R, L210W, T215F or Y and K219Q. Studies on the molecular mechanism of resistance have recently focused on the enhanced primer unblocking activity conferred by these mutations – i.e. the mutant enzyme has increased ability to remove a chain terminator after it has been incorporated. Removal is accomplished by transfer of the chain terminator to an acceptor that may be pyrophosphate or nucleoside di- or triphosphate.The intracellular acceptor substrate for this reaction has not been determined.TAMs are clustered at some distance from the dNTP-binding site in HIV-1 RT, but they are well positioned to interact with a nucleoside triphosphate substrate for the unblocking reaction. RTs containing TAMs have elevated unblocking activity against most chain terminators; however, in culture-based assays for drug susceptibility, the mutant viruses are highly resistant to AZT and only minimally resistant to other nucleosides. Addition of micromolar concentrations of natural dNTPs to the in vitro reaction mixtures results in better agreement with the in vivo assays. In vitro experiments have shown that the removal of chain-terminating nucleotides such as ddAMP or d4TMP is inhibited by micromolar concentrations of the next complementary dNTP, whereas removal of AZTMP is relatively insensitive to this inhibition. The intracellular concentrations of dNTPs in the cells used in culturebased drug susceptibility assays are likely to be high enough to prevent the removal of most chain-terminating nucleotides other than AZTMP. A full understanding of the mechanism of resistance conferred by these mutations will require the definition of the contributions of each residue to nucleotide binding and discrimination, primer unblocking activity, and viral replication fitness.