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.

We have recently reported that the reverse transcriptase (RT) of SIVMNE 170 (170), which is a representative viral clone of the late symptomatic phase of infection with the parental strain, SIVMNE CL8 (CL8), has a largely increased fidelity, compared with the CL8 RT. In the present study, we analyzed the mechanistic alterations of the high fidelity 170 RT variant. First, we found that among several 170 RT mutations, only one, V148I, is solely responsible for the fidelity increase over the CL8 RT. This V148I mutation lies near the Gln-151 residue that we recently found is important to the low fidelity of RT and the binding of incoming dNTPs. Second, we compared dNTP binding affinity (K d ) and catalysis (k pol ) of the CL8 RT and the CL8-V148I RT using pre-steady state kinetic analysis. In this experiment, the high fidelity CL8-V148I RT has largely decreased binding to both correct and incorrect dNTP without altering k pol . The fidelity increase imparted by the V148I mutation is likely because of the major reduction seen in RT binding to dNTPs. This parallels our findings with the Q151N mutant. Third, site-directed mutagenesis targeting amino acid residue 148 has revealed that a valine amino acid at this position is essential to RT infidelity. Based on these findings, we discuss possible structural impacts of residue 148 (and mutations at this site) on the interaction of RT with incoming dNTPs and infer how alterations in these properties may relate to viral replication and fitness.One of the most unique enzymatic properties of lentiviral DNA polymerases (i.e. human and simian immunodeficiency virus (HIV-1 1 and SIV) reverse transcriptases (RT)) is their highly error-prone mode of DNA synthesis (1, 2). The anti-viral immune selection drives the viral diversity that may result from the fast replication of lentiviruses (3-5) and the efficient mutation synthesis catalyzed by lentiviral RTs (2). As recently reported, it is increasingly apparent that infidelity of lentiviral RTs provides the raw material for the viral genomic hypermutation that allows lentiviruses to efficiently evolve and escape from various anti-viral selective pressures (6). Unlike DNA polymerases involved in the cellular genomic replication process, lentiviral RTs lack the 3Ј to 5Ј proofreading nuclease activity, which would normally help prevent mutagenic DNA synthesis. However, this is not the only factor contributing to the low fidelity of lentiviral RTs because RTs of onco-retroviruses lacking the proofreading exonuclease activity, such as murine leukemia virus and avian myeloblastosis virus, have 10 -18-fold higher fidelity than lentiviral RTs (7). This suggests that lentiviral RTs harbor DNA polymerase active sites with unique molecular characteristics responsible for their unfaithful DNA synthesis.Kinetic studies with DNA polymerases suggest that mutation synthesis, which generates a mismatch at the 3Ј end of newly synthesized DNA, interrupts processive DNA polymerization (8). HIV-1 RT is highly efficient in the incorporation of incorrect ...

The causative agent of dental caries in humans, Streptococcus mutans, outcompetes other bacterial species in the oral cavity and causes disease by surviving acidic conditions in dental plaque. We have previously reported that the low-pH survival strategy of S. mutans includes the ability to induce a DNA repair system that appears to involve an enzyme with exonuclease functions (K. Hahn, R. C. Faustoferri, and R. G. Quivey, Jr., Mol. Microbiol 31:1489-1498, 1999). Here, we report overexpression of the S. mutans apurinic/apyrimidinic (AP) endonuclease, Smx, in Escherichia coli; initial characterization of its enzymatic activity; and analysis of an smx mutant strain of S. mutans. Insertional inactivation of the smx gene eliminates the low-pH-inducible exonuclease activity previously reported. In addition, loss of Smx activity renders the mutant strain sensitive to hydrogen peroxide treatment but relatively unaffected by acid-mediated damage or near-UV irradiation. The smx strain of S. mutans was highly sensitive to the combination of iron and hydrogen peroxide, indicating the likely production of hydroxyl radical by Fenton chemistry with concomitant formation of AP sites that are normally processed by the wild-type allele. Smx activity was sufficiently expressed in E. coli to protect an xth mutant strain from the effects of hydrogen peroxide treatment. The data indicate that S. mutans expresses an inducible, class II-like AP endonuclease, encoded by the smx gene, that exhibits exonucleolytic activity and is regulated as part of the acid-adaptive response of the organism. Smx is likely the primary, if not the sole, AP endonuclease induced during growth at low pH values.Streptococcus mutans inhabits dental plaque, where, as a causative agent of dental caries, it creates and survives in an acidic milieu (20). Acidification of plaque is a direct result of the secretion of organic acids, by-products of carbohydrate metabolism by S. mutans and other bacterial inhabitants of the oral cavity. The survival of the microbe in the oral cavity is predicated on its ability to elaborate an acid-adaptive response (30). Since pH values in dental plaque have been reported to be as low as 4.0, the intracellular environment is potentially exposed to acidic conditions (11,12,15), which likely leads to an increased formation of abasic sites in DNA (19). This idea is indirectly supported by our previous observation that the process of acid adaptation in S. mutans also includes expression of a low-pH-inducible apurinic/apyrimidinic (AP) endonuclease (10, 27). These studies suggested that the AP endonuclease activity in S. mutans more closely resembles exonuclease III (Exo III) of Escherichia coli than endonuclease IV (10).Based on this hypothesis, we expected that the gene encoding the AP endonuclease activity in S. mutans would show homology to exoA from Streptococcus pneumoniae and xth from E. coli. In the present study, we have identified and cloned a homologue of the E. coli class II AP endonuclease, exonuclease III, which we have nam...