We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (؊)-2,3-deoxy-3-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a ϳ10-to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k pol values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid -branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.(Ϫ)-2Ј,3Ј-Deoxy-3Ј-thiacytidine (3TC), 3 a deoxycytidine analog reverse transcriptase (RT) inhibitor, has been routinely included in the anti-HIV-1 drug regime (1, 2). During 3TC therapy, two amino acid substitutions at position 184 of RT are sequentially selected, conferring viral resistance to this drug. The M184I mutation is detected earlier, and eventually replaced with the M184V mutation. One initial explanation for the delayed selection of the M184V mutation is the requirement of two nucleotide mutations to develop the final M184V mutation from the wild-type methionine codon (ATG). In contrast, the M184I mutation requires only a single nucleotide mutation from the Met codon, which may result in its early selection during 3TC therapy (3-7).Structural analysis later provided a functional explanation for the instability in vivo of the M184I mutation. The M184I mutation alters the RT interaction with the template nucleotide more significantly than the M184V mutation, leading to more severely decreased processivity, compared with the M184V RT (8). Indeed, the distributive DNA synthesis catalyzed by the M184I RT has been a major explanation for the instability in vivo of this mutation and its rapid transition to the M184V mutation. The long -branched side chains of these two mutations efficiently block the entry of 3TCTP to the active site (8, 9). Interestingly, pre-steady-state kinetic studies of the M184V mutant demonstrated that the M184V mutation only slightly (1-to 2-fold) affects K d (dNTP binding affinity), but not k pol (conformational change/chemical catalysis) steps of HIV-1 RT with normal dNTP substrates, implying that the Val mutation d...
Here, we investigated the pre-steady-state deoxynucleoside triphosphate (dNTP) incorporation kinetics of primate foamy virus (PFV) reverse transcriptase (RT) in comparison with those of HIV-1 and MuLV RTs. PFV RT displayed a drastic reduction in primer extension at low dNTP concentrations where HIV-1 RT remains highly active, indicating a low dNTP binding affinity in the case of PFV RT. Indeed, kinetic analysis showed that, as observed with MuLV RT, PFV RT exhibits ϳ10 to 80 times lower dNTP binding affinity than HIV-1 RT. These three RTs, however, show similar catalytic activities. In conclusion, PFV RT displays mechanistic distinctions in comparison to HIV-1 RT and shares close similarity to MuLV RT.Unlike other retroviruses, foamy viruses (FVs), which have been isolated from various vertebrate animals, are considered nonpathogenic (8, 9, 11). FV naturally targets dividing cells, and FV vector systems have also been shown to efficiently transduce only cells with a mitotic index (13). In contrast, lentiviruses, including human immunodeficiency virus type 1 (HIV-1), uniquely replicate in nondividing cells, such as macrophages and microglia, which are not observed in other groups of retroviruses, including FVs and oncoretroviruses.We recently demonstrated that the unique high affinity of binding of HIV-1 reverse transcriptase (RT) to deoxynucleoside triphosphate (dNTP) substrate contributes to viral infectivity in macrophages that contain very low dNTP pools (ϳ20 to 50 nM), compared to levels in the other natural target cell type, activated CD4 ϩ T cells containing ϳ2 to 5 M dNTP (2). Our follow-up, pre-steady-state kinetic study revealed an unexpected kinetic difference between the RTs of HIV-1 and murine leukemia virus (MuLV). Indeed, HIV-1 RT has ϳ7 to 123 times higher affinity of binding to dNTPs than MuLV RT (12). This and several other studies on reduced-dNTP-binding mutants of HIV-1 RT mimicking MuLV RT (2, 4, 12) suggested that the lower dNTP binding affinity of MuLV RT is still sufficient to support the replication of MuLV. This is because MuLV replicates only in dividing cells containing high cellular dNTP concentrations. An important implication of these studies is that reduction of the dNTP binding affinity of HIV-1 RT can limit viral infectivity to only those cell types containing high dNTP concentrations (2, 3). In this study, we investigated the dNTP incorporation mechanism of primate FV (PFV) RT, using pre-steady-state kinetic analysis, in comparison to those of HIV-1 and MuLV RTs.We hope that this biochemical analysis with PFV RT provides additional supporting evidence that the dNTP binding profile of RT is mechanistically linked with the cell tropism of retroviruses.It was previously demonstrated that RTs with lower dNTP binding affinity, such as MuLV RT, exhibit reduced polymerase/primer extension activity at low dNTP concentrations. In environments with low dNTP concentrations, the RTs with higher dNTP binding affinity, such as HIV-1 RT, still remain active. However, in the case of MuLV RT, dN...
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