The M184V Mutation in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Impairs Rescue of Chain-Terminated DNA Synthesis

Abstract

Nucleoside analog chain terminators such as 3-azido-3-deoxythymidine (AZT) and 2,3-dideoxy-3-thiacytidine (3TC) represent an important class of drugs that are used in the clinic to inhibit the reverse transcriptase (RT) of human immunodeficiency virus type 1. Recent data have suggested that mutant enzymes associated with AZT resistance are capable of removing the chain-terminating residue with much greater efficiency than wild-type RT and this may, in turn, facilitate rescue of DNA synthesis; these experiments were performed using physiological concentrations of pyrophosphate or nucleoside triphosphates, respectively. The present study demonstrates that the M184V mutation, which confers high-level resistance to 3TC, can severely compromise the removal of chain-terminating nucleotides. Pyrophosphorolysis on 3TC-terminated primer strands was not detectable with M184V-containing, as opposed to wild-type, RT, and rescue of AZT-terminated DNA synthesis was significantly decreased with the former enzyme. Thus, mutated RTs associated with resistance to AZT and 3TC possess opposing, and therefore incompatible, phenotypes in this regard. These results are consistent with tissue culture and clinical data showing sustained antiviral effects of AZT in the context of viruses that contain the M184V mutation in the RT-encoding gene.Although considerable progress has been made in the treatment of AIDS, the emergence of mutated variants of human immunodeficiency virus type 1 (HIV-1) resistant to antiviral drugs is a major problem. Resistant breakthrough viruses, and also the persistence of reservoirs of latently infected cells, can limit the success of highly active antiretroviral therapy that involves a combination of potent inhibitors of the virus-encoded reverse transcriptase (RT) and protease enzymes (9,12,13,40). The prolonged clinical use of nucleoside analog chain terminators, e.g., 3Ј-azido-3Ј-deoxythymidine (AZT or zidovudine) and 2Ј,3Ј-dideoxy-3Ј-thiacytidine (3TC or lamivudine) gives rise to resistant viruses that contain mutations in the RT enzyme (4,10,21,22,35,36,37,38). This class of inhibitors competes with natural deoxynucleoside triphosphate (dNTP) pools after being phosphorylated by cellular kinases. DNA synthesis is blocked once the chain terminator is incorporated, since the nucleoside analog lacks a 3Ј-OH group that is required to continue the polymerization process. Mutant enzymes can reduce the probability of incorporation of a chain terminator by lowering the affinity of the latter for the dNTP binding pocket and/or reducing the efficiency of the catalytic step. The crystal structures of HIV-1 RT bound to DNA-DNA with (16) and without (17) an incoming dNTP provide a basis to understand how individual amino acid substitutions might alter the RT structure in the vicinity of the polymerase active site to confer resistance to nucleoside-analog RT inhibitors. Current knowledge with regard to mechanisms of HIV resistance to this class of drugs has recently been reviewed (34).Understanding the mechanisms involv...

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

“…Rescue of Chain-terminated DNA Synthesis-Rescue of DNA synthesis was studied at a single template position using a similar assay as we described earlier (11). The pre-hybridized duplex (8.5 pmol), composed of the aforementioned template and primer strands, was incubated with 25.5 pmol of HIV-1 RT in a buffer containing 50 mM Tris-HCl, pH 7.8, 50 mM NaCl, and 6 mM MgCl 2 followed by the addition 10 M dCTP and 10 M AZT-TP (TriLink BioTechnologies) to generate primer strands terminated with AZT-MP.…”

“…Previous biochemical studies have shown that the incorporated chain terminator can be removed from the primer terminus through phosphorolytic cleavage in the presence of either pyrophosphate (PP i ) (1) or in the presence of ribonucleotide triphosphates (NTPs) (2), which were shown to act as pyrophosphate donors. Increasing evidence suggests that the latter reaction pathway provides an important mechanism for HIV resistance to AZT and, to a certain degree, also to other NRTIs (3)(4)(5)(6)(7)(8)(9)(10)(11).…”

Mutations in the connection domain of HIV-1 reverse transcriptase increase 3′-azido-3′-deoxythymidine resistance

“…2). The pol domain of patient T-6 contained the M184V and L74V substitutions in addition to the TAMs, both of which have been shown to increase AZT sensitivity by decreasing the efficiency of nucleotide excision (12)(13)(14)(15). This result suggested that the cn domain mutations were likely to have been selected during the treatment of patient T-6 and the AZT-sensitizing mutations M184V and L74V were selected later, perhaps in response to treatment with 2Ј,3Ј-dideoxy-3Јthiacytidine (3TC), abacavir and/or dideoxyinosine 2Ј,3Ј-dideoxyinosine (ddI) (SI Table 3).…”

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

“…All pre-steady-state and steady-state experiments were performed as described (1,7,11,39,47). All details, including modifications, are provided in SI Text.…”