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HIV-1 encodes reverse transcriptase (RT) (EC 18.104.22.168 and EC 22.214.171.124), a multifunctional enzyme responsible for converting the single-stranded viral genomic RNA to a double-stranded DNA form, a process that is essential for viral replication and for establishing infection. Although it has many structural similarities to other high-fidelity replicative DNA polymerases, such as a structure resembling a right hand, with fingers, palm and thumb subdomains  and substrate-induced structural changes [2-4], HIV-1 RT is quite error prone with an error rate in the order of 3 × 10−5 mutations/nucleotide/cycle . The relatively high error rate of HIV-1 RT, in conjunction with the rapid replication of the virus in vivo, leads to high genetic variation of the HIV-1 genome in patients, ultimately resulting in immune escape mutants and in drug-resistant variants [6, 7].
Several studies of HIV-1 RT have established a basic model for nucleotide incorporation [3, 8, 9]. The general mechanism of the polymerization process begins with the binding of the template-primer (t/p) to the enzyme (E) to form a binary complex (E: t/p). The incoming deoxyribonucleotide triphosphate (dNTP) is subsequently bound in a loose ‘open’ tertiary complex (E: t/p: dNTP), which undergoes a conformational change into a ‘closed’ tertiary complex ready to undergo the chemical step (E': t/p: dNTP) . A conformational change has been identified as the rate-limiting step of dNTP incorporation [8, 9]. However, kinetic studies demonstrate that the extent to which the conformational change is rate limiting may differ , and it has been specifically suggested that in mismatch extension, conformational change may no longer be the rate-limiting step . During the conformational change, the p66 fingers subdomain closes down on the incoming dNTP, helping to precisely align the 3′-OH of the primer, the α-phosphate of the dNTP and the polymerase active site for chemistry . Upon formation of the new phosphodiester bond, pyrophosphate is released, leaving the t/p extended by one nucleotide and the RT bound such that the primer 3′-end occludes the nucleotide-binding site (Fig. 1) [13, 14]. This complex, referred to as a pretranslocation complex, can be stabilized in vitro by the presence of the pyrophosphate analog, foscarnet. In order to extend the primer with an additional nucleotide, the RT must translocate along the t/p and reposition itself such that the primer 3′-end resides in the priming site [13, 14], leaving the nucleotide-binding site [13, 14] free for nucleotide binding. This post-translocation complex (Fig. 1) can be stabilized in vitro using a 3′-chain-terminated primer in the presence of the next complementary nucleotide.
Figure 1. Graphical representation of the positioning of the RT on matched and mismatched primer templates. Cleavage positions in the RNaseH active site are shown with stars, with the black star indicating the precise position of cleavage for each complex. The positioning of the priming (P) and nucleotide-binding (N) sites of the polymerase active site [13, 14], relative to the t/p are highlighted as green and pink rectangles respectively. The RT forms two complexes with matched primer termini: pretranslocation and post-translocation. Binding to a mismatched primer results in an additional complex (−2).
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Discrimination to reduce error can occur either at nucleotide insertion or following insertion, by promoting removal of an incorrect nucleotide. It is well known that HIV-1 RT does not possess proofreading capability ; thus, error prevention must occur at the point of nucleotide incorporation. The incorporation of an incorrect nucleotide in a DNA strand is the result of two processes: misinsertion and mismatch extension. Misinsertion is the initial insertion of a noncomplementary nucleotide, whereas mismatch extension is the subsequent extension of the mismatched terminus thus formed. HIV-1 RT can efficiently perform both misinsertion and mismatch extension events [16, 17].
Examination of the HIV-1 RT–DNA–dNTP ternary structure implicates a number of amino-acid residues in the formation of the dNTP-binding pocket . Many of the residues lining the dNTP-binding pocket are situated in the highly flexible β3–β4 loop of the fingers subdomain of the p66 subunit (Fig. 2). The β3–β4 loop plays a critical role in orienting and stabilizing the substrate for catalysis [18, 19]. This loop encompasses residues 60–75, which includes Lys70, Lys74, Lys65, Arg72 and Asp67. The three residues of the loop Arg72, Asp67 and Lys65 make contact with the α-, β- and γ-phosphates of the incoming nucleotide, respectively [12, 20-22].
Figure 2. Positioning of Lys66Arg in the β3–β4 loop relative to the primer in the dNTP-binding site. The distance between the Lys66 and the phosphate backbone of the penultimate base pair is labeled with a red line and is measured using the distances tool under the structural analysis menu of Chimera . The ultimate and penultimate nucleotides are indicated. The incoming dNTP is represented as a mesh surface, while the β3–β4 strand is shown in light gray. The relative positions of atoms were obtained from 1rtd .
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Several residues in the β3–β4 loop are implicated in drug resistance as primary mutations, but only a few, including Leu74, Val75 and Lys65, have been identified as playing a role in misinsertion and mismatch extension. Leu74Val mutations have shown up to a six-fold reduction in misinsertion frequency, and a 15- to 26-fold reduction in mismatch extension frequency . Matamoros et al. showed that the mutation Val75Ile increased the mismatch extension fidelity by three-fold while showing no apparent effect on misinsertion fidelity. The authors showed that the increased fidelity is caused by a decrease in nucleotide affinity in the presence of mismatched t/p . Early work from our laboratory investigating the influence of Lys65Arg mutations on overall mutation rates and error specificity indicated a qualitative defect in both misinsertion and mismatch extension, as seen by a reduction in extension products on the gel-based single dNTP exclusion assay . Further investigation into the influence of mutations at Lys65 showed that a Lys65Arg mutation conserves polymerase activity similar to that of the wild-type (WT) enzyme, whereas a nonconservative substitution (i.e. Lys65Ala) results in reduced RT catalytic efficiency (kcat/Km)  as well as decreased affinity for dNTP . The increased fidelity of the Lys65Ala mutation appears to be directly caused by a cumulative effect of a reduction in kpol and increased dNTP Kd in both misinsertion and mismatch extension . Although mutagenesis studies have investigated the role of a number of β3–β4 residues in fidelity [25, 27], a role for Lys66 in RT fidelity has not been investigated so far.
In the primary sequence of RT, Lys66 is in a cluster of three lysines in the fingers subdomain. Unlike Lys64, which points away from the polymerase active site in the 3D structure of a ternary complex, Lys66 lies adjacent to the phosphate of the penultimate primer base/nucleotide (Fig. 2) . This conformation is strictly seen in the presence of t/p and dNTP, while in the absence of bound dNTP, the fingers subdomain is further from the active site [28, 29]. Owing to its location in the vicinity of the dNTP-binding pocket, we hypothesized that Lys66 could play a role in mismatch extension. The significance of this residue is further supported by its broad conservation among RTs of retroviruses and other retroelements . Likewise, Lys66 is conserved in patient isolates. Mutations identified among patient isolates at this position include: Lys66Arg, Lys66Asn, Lys66Thr and Lys66Glu. These mutations are not known to confer drug resistance but rather occur at a low frequency in both drug-naïve and drug-resistant isolates [31, 32].
In the present study, we investigated the potential role of the Lys66 residue in RT fidelity by creating the substitutions Lys66Arg, Lys66Ala, Lys66Asn and Lys66Thr and testing them for both misinsertion efficiency (fins) and mismatch extension efficiency (fext). Our results showed that not only did the Lys66 substitutions display decreased fext, they were also poor at dNTP misinsertion. In addition, drug-susceptibility tests indicated that substitutions at position 66 confer low resistance to nucleotide RT inhibitors such as 3′-azido-3′-deoxythymidine-5′-triphosphate (AZTTP). Furthermore, we showed (using site-specific hydroxyl radical footprinting) that these substitutions do not alter the positioning of HIV-1 RT on the t/p, but rather, similarly to the WT enzyme, their positioning on the t/p substrate is affected in the presence of a mismatched primer terminus.
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In this report, we have shown that Lys66, located in the β3–β4 loop of HIV-1 RT, contributes to mismatch extension fidelity of HIV-1 RT. Perturbing this residue by mutation decreases fext. Interestingly, in addition to mismatch extension fidelity, Lys66 also appears to be important for dNTP insertion fidelity. In addition, we showed that substitutions at this position have little effect on the positioning of the enzyme on the substrate t/p.
In the event that a mispair is created, the enzyme may extend past the mispair, dissociate from the substrate or remain bound as an inactive complex, in which case the enzyme would no longer contribute to the rate of the reaction. Analysis of the t/p-binding kinetic parameters ka (association rate) and kd (dissociation rate) shows that t/p binding does not correlate with mismatch discrimination. A ‘bulkier’ (Arg) or polar uncharged (Asn or Thr) residue shows decreased ka and increased kd for a net slight decrease in affinity indicated by a five- to 50-fold increase in KD. Furthermore, the effect on the KD for matched and mismatched t/p follows similar trends such that no significant differences are seen in affinity of the RT for a matched or a mismatched t/p. As seen previously, a mismatch has little effect on WT t/p binding [27, 32]. Thus, WT RT does not discriminate against a mismatch based on t/p binding and the Lys66 mutations that do increase discrimination do not do so through binding. Although substitutions at position 66 do alter t/p binding, they do not discriminate against the mismatched terminus; rather, they lead to an overall reduction in affinity for the DNA substrate. This was unexpected, as given the proximity of the Lys66 residue to the primer strand, we had hypothesized that discrimination would probably be through altered interaction with the t/p. Furthermore, substituting Lys66 with Arg, Ala, Asn and Thr residues in the structure of a ternary complex of HIV-1 RT showed that nonconservative substitutions caused a greater distortion in the active-site geometry than did the conservative substitutions, and particularly changed the relative position and proximity of the side-chain of residue 66 and the phosphate on the penultimate nucleotide (Fig. 6).
Figure 6. Mapping of the relative positions of Lys66 substitution mutants with respect to the phosphate of the penultimate base pair. Molecular models of residue 66 in comparison with the conservative and nonconservative substitution mutants using the 3° crystal structure of Huang et al. . The β3–β4 strands are shown in yellow, and the incoming dNTP is shown as a mesh surface. The primer strand and the template strand are both labeled, whereas the nucleotides in both strands are color-coded: dA is blue; dG is green; and dC is pink. Each of the nonconservative mutations was introduced into position 66 of model 1rtd using the command line swapaa and the Dunbrack rotamer library . Distances (represented by red lines) were measured from the indicated atom of the residue at position 66 to the phosphate on the primer strand using the distances tool under the structural analysis menu of Chimera .
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Data from our single-nucleotide extension assays showed that while WT and Lys66Arg are efficient in extending from a mismatched primer terminus, the nonconservative mutants showed a five- to six-fold reduction in fext. Analysis of catalytic efficiency (fext) allows one to delineate the influence of the substitution specifically on the mismatch extension, as the role of the mutation in normal catalysis is compensated for. Hence, this reduction in efficiency implicates Lys66 explicitly in mismatch extension fidelity.
The Lys66Arg substitution did not alter the misinsertion fidelity, whereas the nonconservative mutants showed a reduced ability to misinsert. The G:T mispairs are purine:pyrimidine mispairs and are more efficiently formed compared with C:T or T:T mispairs because they are similar to A:T base pairs. All the enzymes were relatively efficient in G:T mispair formation whereas the C:T and T:T mispairs were less tolerated.
Taken together, the data suggest that the Lys66 residue not only facilitates the rather high WT levels of mismatch extensions but also misinsertions. It is likely that the effects on misinsertion and mispair extension are caused by local, active-site distortions, demonstrated by the increases in the distance of the residue side chain to the primer (Fig. 6) for the substitution mutants.
Although there are subtle, and statistically significant, differences between the positions of the WT and nonconservative Lys66 substituted enzymes on the mismatched primer terminus, these differences do not account for the differences in fidelity that were observed. The extent to which the enzyme is found in a −19 (−2) additional pretranslocation position differs significantly for WT RT and the Lys66Ala, Asn and Thr mutants (Fig. 5C, right panel). In the context of a mismatched t/p, more of the WT enzyme shifts backwards to a −19 (−2) position compared with the nonconservative mutants. This suggests that upon encountering an incorrect base pair at the primer 3′-terminus the enzyme could potentially realign its active site by sliding backwards on the t/p. Likewise, there is bias in the percentage of complexes found at position −17 on the mismatched t/p, with more of the nonconservative mutant complexes found at this position. Interestingly, although the enzymes in complexes at position −17 are positioned such that they can readily accept an incoming nucleotide, this increase in post-translocation complexes does not correlate with an increase in extension from a mismatched t/p for Lys66Ala, Asn and Thr. This discrepancy between t/p positioning and mismatch extension efficiency was also described previously for a Lys65Ala mutant .
HIV-1 RT has evolved the ability to extend mismatches and misinsertions efficiently. None of the amino-acid residues that are responsible for this property have been delineated. For dNTP misinsertion, a number of residues that line the dNTP-binding pocket, including Lys65, Thr115 and Met184, have been shown to impact RT fidelity [26, 35, 36]. Residues affecting the fidelity of RT are not limited to those situated in the polymerase active site. Several substitutions at residues that do not directly line the active site, but yet impinge upon it via interaction with template or primer, such as Met230Ile (in the primer grip)  and Glu89Gly (at position −2 on the template) [23, 38] have also been shown to bring about increases in fidelity. Similarly, as seen from our data, substitutions at the Lys66 residue, which appears poised to contact the penultimate nucleotide at the primer 3′-terminus, led to enhanced fidelity of misinsertion. In addition to the impact on mispair extension fidelity that we predicted based on the proximity of Lys66 to the primer terminus, the Lys66 substitutions also enhanced the fidelity of misinsertion. Previously, through substitution of Lys65, which contacts the gamma-phosphate of the incoming nucleotide, it was demonstrated that this residue also has a role in both aspects of fidelity. Although the Lys65 and Lys66 residues are adjacent to each other in the β3–β4 loop, the similarity of the effect of substitutions of these residues on fidelity was unexpected, as their orientation, as shown in the crystal structure, is quite different from each other.
In an attempt to investigate whether the similarity between Lys66 and Lys65 would extend to drug susceptibilities, we performed both gel-based and filter-based RT assays. In addition, Lys65Arg has also been identified as the primary mutation selected for by TFV [39, 40] and has been shown to confer cross-resistance to other inhibitors, including 2′-3′-didehydro-2′-3′-dideoxythymidine (d4T) in cell-based assays  and its triphosphate form, d4TTP in cell-free assays . Here we showed that the nonconservative Lys66 mutants showed phenotypes that are quite different from the WT but not as profound as the resistance profiles seen with the Lys65 mutants. While the Lys65Arg mutation has been widely studied in drug resistance, the drug-resistance effects of mutations at position 66 have not been tested. However, it is of interest that mutations at position 66 (which rarely occur in the virus) lead to decreases in drug susceptibility, suggesting that the high conservation of this residue is important for viral fitness. The differences in mutation frequencies observed when substitutions of WT residues are created at positions 65 and 66 may reflect the degree of resistance conferred by these substitutions; thus, although the Lys65 substitutions have a greater effect on enzymatic activity than the Lys66 substitutions, they also lead to higher levels of drug resistance.