HIV-1 reverse transcriptase (RT) is a highly error prone DNA polymerase. We assessed whether the ability of RT to bind nucleotide substrates affects viral mutagenesis. Structural modeling predicts that the V148 and Q151 residues influence the interaction between RT and the incoming dNTP. When we introduce either a V148I or Q151N mutation, RT fidelity increases 8.7- or 13-fold, respectively, as measured by the M13 lacZalpha forward mutation assay. Interestingly, pre-steady state kinetic studies demonstrated that these mutations do not alter polymerase fidelity during the first step of mutation synthesis, misincorporation. Rather, the V148I and Q151N mutations alter RT fidelity by weakening the ability of the polymerase to complete mismatch extension, the second step of mutation synthesis. While both these mutations minimally affect the binding of RT (K(D)) to a mismatched template-primer complex (T/P), these mutant RTs are significantly impaired in their ability to bind (K(d)) and chemically incorporate (k(pol)) nucleotide substrate onto a mismatched T/P. These differences in binding and catalysis translate into 24- and 15.9-fold increase in mismatch extension fidelity for the V148I and Q151N RT mutants, respectively. Finally, we employed a cell-based pseudotyped HIV-1 mutation assay to determine whether changes in these dNTP binding residues alter RT fidelity in vivo. We found that the V148I and Q151N mutant viruses had 3.8- and 5.7-fold higher fidelities than wild-type viruses, respectively, indicating that the molecular interaction between HIV-1 RT and the dNTP substrate contributes to viral mutagenesis.
We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K d ) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k pol ). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K d and k pol values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.Retroviruses encode a versatile DNA polymerase called reverse transcriptase (RT).1 The function of RT is to synthesize linear double-stranded proviral DNAs from single-stranded positive sense viral RNA genomes during viral replication. In order to catalyze this process, RTs perform several distinct enzymatic reactions including RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, strand transfer, and RNase H cleavage (1). The DNA polymerase activity of RTs has been targeted, using various types of RT inhibitors such as nucleoside substrate-like compounds (i.e. azidothymidine (AZT) and didanosine (ddI)) (2), as a means to reduce viral replication in infected individuals. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) uniquely infect terminally differentiated/nondividing cells (i.e. macrophages) as well as dividing cells (i.e. activated CD4ϩ T cells), whereas oncoretroviruses such as murine leukemia virus (MuLV) productively replicate mainly in dividing cells (3,4). Numerous studies have reported that actively dividing cells have higher cellular deoxynucleotide triphosphate (dNTP) concentrations than nondividing cells (5). Recently, it was reported that the cellular dNTP concentration of human macrophages (ϳ40 nM) is ϳ100 times lower than that of dividing CD4 ϩ T cells (ϳ5 M) (6). Therefore, RTs ...
It has previously been reported that mutations in the Gln 151 residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln 151 in highly error prone DNA synthesis by HIV-1 RT. Using our Q151N high fidelity mutant, which is structurally altered in its ability to interact with the 3-OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity (K D ) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln 151 residue is not involved in the formation of the binary complex (RT⅐template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln3 Asn, the maximum rate of dNTP incorporation (k pol ) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (K d ) was diminished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. Presumably, the fidelity increase observed during the steady state is explained by this defect in Q151N binding to incorrect dNTP. In wild type RT, residue Gln 151 is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln 151 residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln 151 is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.It is becoming more apparent that many organisms employ multiple DNA polymerases to replicate their genomes. Some of these DNA polymerases are specifically involved in error prone DNA synthesis required for either spontaneous mutagenesis or bypassing DNA damage (1-3). Recent biochemical studies of DNA polymerases and and their bacterial UmuC/DinB homologs (4 -6) show that these are actually very low fidelity polymerases. It is possible that these organisms have evolved functionally diverse DNA polymerases for the different activities of genomic replication and mutagenesis. As demonstrated in a series of kinetic experiments with various DNA polymerases, it is clear that the ability to incorporate incorrect dNTPs 1 affects the efficiency of DNA synthesis (7). In other words, low fidelity and poor ability to discriminate between correct and incorrect dNTPs are detrimental to efficient DNA polymerization, which is likely essential for chromosomal DNA replication. The fact that efficient DNA synthesis and error prone DNA synthesis are kinetically at odds with one another may explain why possession of separat...
Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a putative source of the genomic hypermutation that promotes rapid evolution of HIV-1. To understand the molecular strategies that create a highly mutagenic DNA polymerase active site in HIV-1 RT, we investigated the roles of four residues in the beta 8-alpha E loop. Gln151, which interacts with the sugar of the incoming dNTP, and Lys154, which interacts with the template, yielded site-directed mutants with increased fidelity, suggesting that these residues are directly involved in the mutagenic architecture of the active site. Mutations at Gln151 and Lys154 also reduced processivity. Q151N RT showed enhanced ability to discriminate between TTP and AZT triphosphate, consistent with the observation that the Q151M mutation confers AZT resistance in vivo. Mutations at Gly152 greatly decreased RT activity; molecular modeling suggests that Gly152 is critical for the proper geometric alignment that permits base-pairing of the incoming dNTP with the template. Mutations at Trp153 reduced the expression level, and presumably the stability, of RT proteins in bacteria. These observations support the conclusion that interactions of active site residues in the beta 8-alpha E loop with incoming dNTPs and the template are determinants of the accuracy, processivity, and substrate selectivity of HIV-1 RT.
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