Combinations of antiretroviral drugs that prevent or delay the appearance of drug-resistant human immunodeficiency virus-type 1 (HIV-1) mutants are urgently required. Mutants resistant to 3'-azidothymidine (AZT, zidovudine) became phenotypically sensitive in vitro by mutation of residue 184 of viral reverse transcriptase to valine, which also induced resistance to (-)2'-deoxy-3'-thiacytidine (3TC). Furthermore, AZT-3TC coresistance was not observed during extensive in vitro selection with both drugs. In vivo AZT-3TC combination therapy resulted in a markedly greater decreased in serum HIV-1 RNA concentrations than treatment with AZT alone, even though valine-184 mutants rapidly emerged. Most samples assessed from the combination group remained AZT sensitive at 24 weeks of therapy, consistent with in vitro mutation studies.

HIV reverse transcriptase (RT) is one of the main targets for the action of anti-AIDS drugs. Many of these drugs [e.g., 3-azido-3-deoxythymidine (AZT) and 2,3-dideoxyinosine (ddI)] are analogues of the nucleoside substrates used by the HIV RT. One of the main problems in anti-HIV therapy is the selection of a mutant virus with reduced drug sensitivity. Drug resistance in HIV is generated for nucleoside analogue inhibitors by mutations in HIV RT. However, most of these mutations are situated some distance from the polymerase active site, giving rise to questions concerning the mechanism of resistance. To understand the possible structural bases for this, the crystal structures of AZT-and ddI-resistant RTs have been determined. For the ddI-resistant RT with a mutation at residue 74, no significant conformational changes were observed for the p66 subunit. In contrast, for the AZT-resistant RT (RTMC) bearing four mutations, two of these (at 215 and 219) give rise to a conformational change that propagates to the active site aspartate residues. Thus, these drug resistance mutations produce an effect at the RT polymerase site mediated simply by the protein. It is likely that such long-range effects could represent a common mechanism for generating drug resistance in other systems.Nucleoside analogue inhibitors (NIs) such as 3Ј-azido-3Ј-deoxythymidine (AZT) and 2Ј,3Ј-dideoxyinosine (ddI) are used widely in the treatment of HIV infection. They act by competing with natural substrates at the HIV reverse transcriptase (RT) polymerase active site, leading to incorporation and termination of the DNA chain. Treatment of AIDS patients with NIs selects for HIV strains containing mutations in the RT, which represents a continuing problem in combating the disease, despite the encouraging results from combination drug trials (1). Resistance to AZT is associated with a series of mutations. For instance, the mutations D67N, K70R, T215F, and K219Q give a three-fold increase in K i for AZT-triphosphate; the resultant enzyme denoted RTMC is investigated here (2). For ddI, resistance can be conferred by a single mutation at position 74 (3). Surprisingly, most of the NI resistance mutations in RT are positioned some distance from the polymerase active site in the p66 subunit. In the three-dimensional structure of HIV-1 RT (4-7), residues 215 and 219 are Ϸ10Å from the conserved active site aspartate residues 110, 185, and 186 whereas residues 67, 70, and 74 are in a different domain, Ͼ20 Å away (Fig. 1). The analysis of the effects of the mutations in RTMC on AZT resistance suggests that the interactions between these mutations are complex, ranging from compensatory to synergistic, although residue 215 stands out as having a pivotal role (37). In contrast to NIs, resistance mutations associated with the nonnucleoside RT inhibitors (NNI) cluster around the NNI binding pocket and presumably cause resistance through distortion of this ligand site (6,8,9). In the absence of structural data for NI-resistant RTs, various mechanisms hav...

Six structures of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing combinations of resistance mutations for zidovudine (AZT) (M41L and T215Y) or lamivudine (M184V) have been determined as inhibitor complexes. Minimal conformational changes in the polymerase or nonnucleoside RT inhibitor sites compared to the mutant RTMC (D67N, K70R, T215F, and K219N) are observed, indicating that such changes may occur only with certain combinations of mutations. Model building M41L and T215Y into HIV-1 RT-DNA and docking in ATP that is utilized in the pyrophosphorolysis reaction for AZT resistance indicates that some conformational rearrangement appears necessary in RT for ATP to interact simultaneously with the M41L and T215Y mutations.Codon 215 mutations in combination with M41L in reverse transcriptase (RT) confer high-level zidovudine (AZT) resistance on human immunodeficiency virus (HIV) (9, 11). M184V gives resistance to lamivudine (3TC) (20, 23) and restores AZT sensitivity to codon 41 and 215 mutations (12). X-ray crystallographic results indicate that residues 41 and 215 in HIV-1 RT are distal to the deoxynucleoside triphosphate binding site (6, 7, 10, 16), and ideas as to how AZT resistance is induced include template rearrangement (3) and conformational changes propagated from residues 215 and 219 for the mutant RTMC (D67N, K70R, T215F, and K219N) (17). Biochemical studies have shown a mechanism of AZT resistance involving AZTMP removal from the blocked primer strand in a pyrophosphate-or ATP-dependent pyrophosphorolysis reaction enhanced by RTMC and other mutant RTs (1,2,13,14). Model building studies suggest that T215Y might promote ATP binding, the presumed physiological acceptor for the pyrophosphorolysis reaction (2). To investigate further the effects of AZT and 3TC drug resistance mutations on the conformational states of HIV-1 RT, structures of a series of six AZT-and/or 3TC-resistant RTs mainly containing combinations of M41L, T215Y, and M184V mutations were determined in the absence of oligonucleotide or deoxynucleoside triphosphate substrates to assess whether underlying baseline structural rearrangements are induced by these mutations in a way analogous to that previously observed for RTMC.

Cross-resistance was not commonly observed in this lamivudine-treated cohort. M184V per se is not expected to compromise subsequent treatment with NRTI such as didanosine-stavudine or combinations containing abacavir.