INTRODUCTION

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Human immunodeficiency virus type 1 (HIV-1) infections are characterized by a high degree of genetic variation, which inevitably leads to facile generation of drug-resistant variants during treatment (16, 26). To date, five nucleoside analogs which target reverse transcriptase (RT) have been approved for treating infected individuals. Since monotherapies with each drug culminate in the emergence of resistant mutants, combinations of two or more drugs are preferred (8, 15, 22). One such combination is 2',3'-dideoxy-3'-azidothymidine (AZT) with either 2',3'-dideoxycytidine (ddC) or 2',3'-dideoxyinosine (ddI) in simultaneous or sequential therapy (29, 30). However, the emergence of variant viruses that are resistant to one of the drugs in the regimen, as well as those that are resistant to multiple nucleoside analogs (29, 30), has been reported. Chronological studies of viral variants over an extended period (up to 48 months) revealed the appearance of a series of five mutations (13, 30). Of these, the Q151M mutation appears first and alone is sufficient to confer multidrug resistance to HIV-1, exhibiting a 10-fold increase in resistance to AZT, a 20-fold increase in resistance to ddC, and a 5-fold increase in resistance to ddI in cell culture virus replication assays (30). Subsequently, mutations appearing at four other sites (A62V, V75I, F77L, and F116Y) further increase the level of drug resistance. Viruses containing all five mutations exhibit up to a 320-fold increase in resistance to AZT, up to a 45-fold increase in resistance to ddC, and up to a 40-fold increase in resistance to ddI in cell culture (31). When recombinant purified RTs containing the Q151M alteration alone or the complete set of five mutations were studied via biochemical assays, they exhibited high-level resistance to ddATP, ddCTP, ddGTP, ddTTP, and AZT triphosphate (33, 34).

Mutations in RT that result in nucleoside analog resistance are thought to do so by decreasing the ability to bind and utilize a nucleoside analog while retaining the ability to utilize deoxynucleoside triphosphate (dNTP) substrates in the polymerization reaction (19, 33, 34). If these mutations affect the geometry of the dNTP-binding pocket to preferentially utilize the normal dNTP substrate, it is conceivable that the mutations may also impart an ability to favor the insertion of correctly base-paired dNTPs over incorrectly base-paired dNTPs. Indeed, it has recently been shown that the 2',3'-dideoxy-3'-thiacytidine-resistant M184V RT mutant (11, 21, 35) and the multidrug-resistant E89G HIV-1 RT mutant (9, 27a) exhibit a significant increase in nucleotide insertion fidelity over that of the wild-type enzyme. Mutagenesis and biochemical studies have implicated residue Q151 of HIV-1 RT in dNTP-binding function (28). Furthermore, the Q151 residue lies within the LPQG motif that is conserved in RTs (23). The three-dimensional structure of HIV-1 RT complexed with double-stranded DNA reveals that the Q151 residue is in a position to interact with the first nucleotide of the single-stranded template (30). Additionally, the high-resolution X-ray structure of a fragment of the Moloney murine leukemia virus RT reveals that the residue Q190, which is analogous to Q151 of HIV-1 RT, interacts with the templating base and the incoming dNTP (12), suggesting that it may determine the fidelity of DNA synthesis. Three of the remaining four mutations that occur in AZT-ddI or AZT-ddC combination therapy also map to the DNA-protein interface, including residue 116, which may be involved in dNTP binding (30). Therefore, we have examined the effect of the Q151M mutation alone as well as in combination with the remaining four mutations (VILYM [see below]) on the overall fidelity of DNA synthesis by HIV-1 RT by using an M13-based forward mutation assay previously described by Kunkel and coworkers (3, 27). Our results show that the mutations cause a small change in the overall mutation frequency, but important differences in mutational specificities were evident when the three enzymes were compared.

(The data in this paper are from a thesis to be submitted by L. F. Rezende in partial fulfillment of the requirements for a Ph.D. in the Sue Golding Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University.)

	MATERIALS AND METHODS

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Phage DNA and bacterial strains. The gapped-duplex DNA substrate was derived from the bacteriophage M13mp2. The M13 phage was grown in Escherichia coli NR9099 [(pro-lac) thi ara recA56/F' (proAB lacI_qZM15)] for the preparation of both single-stranded and replicative-form DNAs. E. coli MC1061 [hsdR hsdM⁺ araD  (ara leu) (lacIPOZY) galU galK strA] was used for electroporation of the products of the fill-in reaction to produce phage. E. coli CSH50 [(pro-lac) thi ara strA/F' (proAB lacI_qZM15 traD36)] was the -complementation strain used to score for the mutant phage.

Enzymes. Bacterial expression and purification of homodimers of wild-type HIV-1_BH10 RT, the Q151M mutant RT, and the VILYM (containing the five mutations A62V, V75I, F77L, F116Y, and Q151M) mutant RT have been described by Ueno et al. (34). The specific activities of these RTs were found to be 500 U/mg for the wild-type enzyme, 833 U/mg for the Q151M RT, and 625 U/mg for the VILYM RT. One unit is defined as the amount of enzyme required to incorporate 1 nmol of dTMP into DNA on a poly(rA) · oligo(dT) template-primer at 37°C in 10 min.

Determination of forward mutation frequency. The M13mp2 duplex DNA containing a single-stranded gap of 361 nucleotides (including the lacZ upstream regulatory sequences and the first 107 nucleotides of the open reading frame) was prepared as previously described (6) and used as a template-primer to perform the DNA synthesis reactions in vitro with purified wild-type and mutant RTs. DNA synthesis reactions were performed in a total volume of 25 µl containing 75 mM Tris-Cl (pH 8.0), 80 mM KCl, 6 mM MgCl₂, 10 mM dithiothreitol, 500 µM (each) dATP, dCTP, dGTP, and dTTP (Boehringer Mannheim, Indianapolis, Ind.), 59 ng of gapped-duplex DNA, and 0.7 to 1.3 U of purified RT for 1 h at 37°C. Complete synthesis of the gapped region was confirmed by gel electrophoresis. Two independent fill-in reactions were performed with each enzyme.

Sequencing of phage DNA. Single-stranded DNA was prepared from mutant plaques as described by Bebenek and Kunkel (6). Briefly, 50 µl of plaque supernatant and 200 µl of an overnight culture of E. coli CSH50 were added to 1.8 ml of 2xYT broth and incubated at 37°C for approximately 15 h. Culture supernatants were collected, and the phage were precipitated with one-fourth the volume of 15% polyethylene glycol-2.5 M NaCl for 6 min at 4°C. Phage were decoated by incubation with 50 µg of proteinase K for 30 min, and single-stranded DNA was precipitated by treatment with 1/10 volume of 5% hexadecyl trimethylammonium bromide in 0.5 mM NaCl for 10 min followed by centrifugation at 12,000 × g. The DNA pellets were resuspended in 1.2 M NaCl, and finally the single-stranded phage DNA was precipitated with ethanol.

Calculation of error rates. Error rates were calculated as described by Bebenek and Kunkel (6). Since two fill-in reactions were performed for each enzyme and the mixtures were separately electroporated, we calculated the mutation frequencies separately and derived the mean values and standard errors. The mean mutational frequency was corrected by subtracting the background mutational frequency. The corrected mean mutational frequency is multiplied by the percentage of all mutations represented by the particular class of mutations (e.g., base substitutions). This number was divided by 0.6 (the likelihood of expression of the newly synthesized strand in E. coli) and then divided by the total number of sites where this class of mutations can be detected within our 361-base target (6).

Statistical analysis. Differences between mutation frequencies were calculated by using the unpaired t test. Differences in the proportions of errors at specific sites were calculated by using the two-tailed Fisher's exact test.

	RESULTS

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Mutational frequencies of wild-type and multidrug-resistant RTs. We used the M13-based forward assay to assess the overall fidelity of the wild-type, Q151M, and VILYM (A62V/V75I/F77L/Q151M/F116Y) RTs. This assay allows the measurement of both the overall fidelity and the rates of specific mutations by each mutant RT. The M13 gapped-duplex substrate contained a single-stranded gap over the regulatory sequences and some of the coding region of the lacZ gene. All three enzymes were capable of synthesizing DNA, resulting in gap closure as determined by agarose gel electrophoresis (Fig. 1).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Ethidium bromide-stained agarose gel showing synthesis of DNA across the gapped-duplex DNA substrate with recombinant, purified HIV-1 RT. Complete synthesis of DNA across the gapped-duplex DNA substrate was confirmed on a 0.8% agarose gel run at 25 V for 20 h. The synthesis is indicated by the gel mobility shift observed between the initial gapped-duplex DNA (no RT) and the products obtained after filling in by wild-type, Q151M, and the VILYM RTs. ds, double stranded.

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Spectra of mutations by wild-type and multidrug-resistant HIV-1 RTs. In order to determine the specificity of mutations generated during the gap fill-in DNA synthesis reaction, M13 templates from 117 plaques representing the mutations induced by the wild-type enzyme, 103 plaques representing the errors by the Q151M RT, and 101 plaques representing those by the VILYM RT were randomly selected from each independent electroporation for sequence determination. A comparison of the mutational spectra of the wild-type and Q151M RTs is shown in the top panel of Fig. 2, while that for wild-type and VILYM RTs is shown in the bottom panel of Fig. 2. It is readily apparent from these mutational spectra that there are differences in the error specificities of the wild-type and the mutant RTs. An examination of the mutation spectrum generated by the wild-type RT reveals four prominent mutational hot spots (consisting of at least six errors at a given site), each occurring at runs of nucleotides. Of these, three are substitution hot spots located at the run of Ts at positions 36 to 34, the run of Gs at positions 88 to 90, and the first of two Ts at positions 112 to 113. The fourth is a frameshift hot spot located in the run of Ts at positions 137 to 139. Three milder hot spots (consisting of three errors at a given site) are also observed for wild-type HIV-1 RT: one at the first of two Gs at position 29, a second at the first T of the pair at position 121, and a third at the last G in the stretch of sequence shown (Fig. 2).

View larger version (78K): [in this window] [in a new window]   FIG. 2.   Spectra of mutations generated by wild-type and nucleoside analog-resistant HIV-1 RTs. The wild-type lacZ sequence is represented, with the corresponding nucleotide positions indicated below the sequence. Mutations created by the wild-type HIV-1 RT are shown above the lacZ sequence, and those generated by one of the mutant RTs are shown below the sequence. Base substitutions are noted directly above or below the lacZ sequence and represent the nucleotide in the mutated template. Frameshift mutations are indicated with an upright triangle for single-base deletions and with an inverted triangle for single-base insertions. Since it cannot be determined exactly which base in a run of nucleotides has been deleted or inserted, the frameshift events are indicated in the middle of the run. (Top) Comparison of the spectra of mutations generated by the wild-type and Q151M variant HIV-1 RTs. (Bottom) Comparison of wild-type and VILYM RTs. In addition to the mutations shown here, two templates generated by the VILYM RT contained large deletions (see text).

	DISCUSSION

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Fidelity studies of HIV-1 RT, using either gel-based primer extension assays or the gap-filling assay with the lacZ gene, have shown that this polymerase has a high error rate (24, 27). More recent studies on the nucleoside analog-resistant RTs, such as those with M184V or E89G alterations, have shown an increase of fidelity (9, 11, 21, 27a, 35). However, those studies employed a gel-based primer extension assay, which allows one to specifically measure the efficiency of misinsertion and does not allow detection of changes in the efficiency of mispair extension or the generation of frameshift mutations. Furthermore, the assay allows for the measurement of misinsertion fidelity under conditions where the enzyme does not have the option to make the correct insertion. In this study, we have used the M13-based forward mutation assay to assess the overall fidelities and mutational specificities of mutant RTs which are parts of variant viruses seen in a subset of patients receiving long-term combination therapy with AZT and ddI or with AZT and ddC. We were able to show that while no significant difference in overall fidelity was observed, important differences in the mutational specificity were seen when wild-type and mutant enzymes were compared.

By using the M13-based gap-filling in vitro mutation assay, the mutation frequency of HIV-1_NY5 RT has been estimated to be 400 × 10⁴ nucleotides (27). In this study, however, the wild-type HIV-1_BH10 RT had a mutation frequency of 64 × 10⁴. Thus, the wild-type RT used here displays an approximately 6.5-fold-lower mutation frequency in vitro than that reported for RT derived from HIV-1_NY5 (27). A second difference between the previous in vitro study and this study is the proportion of frameshift mutations. The ratios of substitutions to frameshifts observed here were 4.5:1, 6.25:1, and 3.3:1 for wild-type, Q151M, and VILYM RTs, respectively. These ratios contrast with a 1:1 ratio observed previously by Bebenek et al. (3). These differences cannot be explained merely by the fact that the strains of HIV used in these two studies are different. In recent studies, the HIV-1_NY5 RT has also been shown to display decreased affinity for template (compared with HxB2-derived RT), as revealed by a 279-fold decrease in the K_m for the template-primer (2) and a greater-than-200-fold increase in the K_off on poly(rA) · oligo(dT) template-primers (14). Poor affinity of RT for the template-primer is thought to promote poorer processivity (1), which in turn can decrease the fidelity of the polymerase (32). Subsequent studies by Bebenek et al. using HIV-1_HxB2 RT showed lower mutation frequencies (200 × 10⁴ to 210 × 10⁴) (5). We have also performed the forward assay with HIV-1_HxB2 RT and have found a mutation frequency of 97 × 10⁴, similar to that we have found for RT from HIV-1_BH10 reported here (24a). When comparing the wild-type and nucleoside analog-resistant RTs from this study, the increases in fidelity are found to be small (1.1-fold for the single mutant and 2.0-fold for the quintuple mutant) and do not reach the difference seen between RTs of different strains, making the difference seem negligible. The fidelities of Moloney murine leukemia virus RT and avian myeloblastosis virus RT have been studied previously by using this assay (27), and the mutation frequencies were found to be 22 × 10⁴ and 42 × 10⁴, respectively, which are similar to the mutation frequencies determined for HIV-1 RT in this study.

Interestingly, differences between the mutational specificities of the wild-type and mutant enzymes were revealed by our studies. It is well documented that the context of a nucleotide site affects the mutation frequency (20, 25). Our data suggest that the overall structure of the enzyme template-primer-binding pocket may also affect which portions of the template are susceptible to mutation. The Q151M RT showed the generation of new hot spots neither previously reported for HIV-1 RT (3, 10, 17, 18) nor observed in our laboratory (24a). Three of these sites (positions 40, 42, and 144) showed statistically significant increases in proportions of mutations (P < 0.05 by the two-tailed Fisher's exact test), with one site (position 42) showing an increase at a more rigorous degree of significance (P < 0.002 by the two-tailed Fisher's exact test) that is necessary to avoid false positives due to type I error. The Q151 residue in HIV-1 RT maps to the loop between the E and 8 region of the RT, near the single-stranded DNA of the template base (30), and is proposed to make contact with the deoxyribose backbone of the templated base (28). The amino acid change from a glutamine to a methionine reduces the size of the side chain. This may add flexibility to the template base, changing the context specificity in which mutations are made. The Q151 residue has been shown to be involved in dNTP binding (28), which may also allow for changes in the nucleotide base pairing allowed by the enzyme. The most striking change in the multiply mutated nucleoside analog-resistant RT is the decrease in misalignment-mediated substitutions and the decrease in mutations at two common mutation sites, 36 and 89. The presence of multiple mutations in this RT, however, precludes any speculations on mechanistic explanations for this phenotype.

We and others (9, 11, 21, 27a, 35) have previously reported coexistence of nucleoside analog resistance with an increase in dNTP insertion fidelity. In this study, however, we find only modest increases in fidelity with another class of nucleoside analog-resistant mutants. The in vivo effects of the alteration of mutational specificity by these RT mutations in vitro need to be assessed. It is conceivable that dramatic changes in the mutational specificity of RT could alter the course of evolution of the HIV-1 quasispecies in infected individuals. It is, however, unclear whether the small changes in error specificity observed here will have a major impact on viral variation. This remains to be assessed via a single-cycle infection assay.