©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Arginine 72 of HIV-1 Reverse Transcriptase
CATALYTIC ROLE AND INHIBITOR SENSITIVITY (*)

(Received for publication, March 27, 1995; and in revised form, June 21, 1995)

Stefanos G. Sarafianos Virendra N. Pandey Neerja Kaushik Mukund J. Modak (§)

From the Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey 07103

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In order to determine the catalytic role of Arg of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the k of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in k was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PP(i). Arg is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.


INTRODUCTION

HIV-1 reverse transcriptase (RT) (^1)is a multifunctional heterodimeric enzyme that catalyzes the incorporation of deoxyribonucleotides using both RNA and DNA templates. It is processed initially from the pol gene product as a 66-kDa polypeptide(2, 3, 4) . Subsequent proteolytic cleavage of the p66/p66 homodimer yields the p66/p51 heterodimer(2, 3, 4) . HIV-1 RT shares some common features with other DNA polymerases, including a large cleft that accommodates the TP, formed by structurally similar three-dimensional motifs which have been called palm, fingers, and thumb(5, 6) . In the cleft of the enzyme resides the ``catalytic triad'' of three acidic residues. These residues are Asp, Asp, and Asp of the p66 subunit of HIV-1 RT(7) . They are proposed to bind to the divalent metal ion(s) required for the polymerase activity(5, 7) , thereby, indirectly affecting the binding of the dNTPbulletMg complex. In addition, other neighboring residues are expected to participate in other vital processes, such as dNTP binding, pyrophosphate product removal, and translocation along the template primer. To our knowledge, with the exception of Q151(16), no other residue in HIV-1 RT has been experimentally shown to participate either in the dNTP binding function or in the PP(i) removal function. The observation that resistance to a number of nucleoside-analog inhibitors in RT is associated with mutation(s) of some residue(s) in the region spanning 65-74 has suggested a possible influence of this region in the dNTP binding function. Furthermore, an antibody against an oligopeptide containing the amino acid sequence of this region inhibited the polymerase activity competitively, with respect to dNTP(8) . However, the activities of most of these mutants were either unaffected or only slightly reduced(9) . In the case of L74V the K for ddATP was found to be increased but no change in Kfor dATP was apparent(10) , implying no direct role for Leu in dNTP binding. Recent studies have suggested that this region could be involved in the binding of template-strand binding (11) rather than of dNTP. Therefore, a considerable degree of uncertainty exists regarding the role of residues in this region.

We have chosen Arg as a target for in vitro site-directed mutagenesis to investigate the role of this region in general and that of Arg in particular. The choice was based on the fact that our comparative modeling studies of the Klenow fragment (KF) and HIV-1 RT have suggested a spatially equivalent location of Arg of RT with that of Lys of KF(12) . Earlier studies on Lys of KF had implicated it in both the dNTP binding function and in the translocation of enzyme across the template(13) . The present work provides experimental evidence regarding the role of Arg, using the R72A and R72K mutants of HIV-1 RT. Furthermore, in order to determine the subunit specificity, if any, we examined the properties of reconstituted hybrid heterodimers, containing mutations only in one subunit (p66/p51 and p66/p51). Our results show that the mutational effects of Arg are expressed through the p66 (and not the p51) subunit. In addition, Arg does not appear to be required for dNTP or DNA binding. The severe impairment in the processive mode of DNA synthesis by R72A together with the inability to catalyze pyrophosphorolysis and the complete resistance to phosphonoformate (a PP(i) analog), strongly implicate Arg in the PP(i) binding/removal function of HIV-1 RT.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes and high performance liquid chromatography purified dNTPs were from Boehringer Mannheim. DNA sequencing reagents were from U. S. Biochemical Corp. Synthetic TPs were from Pharmacia Biotech Inc., and P-labeled dNTPs and ATP were products of Dupont NEN. Oligonucleotides were purchased from Midland Certified Reagent, Dallas, TX.

Methods

Reverse Transcriptase Activity Assay

RT activity was assayed using a standard method(14, 15) . Assay solution contained 200 nM homopolymeric TP, 0.02 mM [^3H]dNTP (1 µCi/assay), 80 mM NaCl, 1 mM DTT, 50 mM HEPES, pH 8.0, and 5 mM MgCl(2). Typically, an aliquot containing approximately 4 nM of WT or 12 nM of mutant enzyme in a final volume of 100 µl was incubated at 37 °C. The reaction was initiated by the addition of MgCl(2), terminated by 5% ice-cold trichloroacetic acid, filtered with Whatman GF/B filters, and counted for radioactivity(17) .

Steady State Kinetics of Polymerization

The kinetic studies were carried out as described by Majumdar et al.(18) using homopolymeric poly(rA) bullet (dT) poly(rC)bullet(dG), or poly(dC)bullet(dG) as TPs and dTTP or dGTP as nucleotide substrates. Km values for dNTP were determined at saturating TP concentrations (200 nM), whereas K values for DNA at saturating dNTP concentrations (0.15 mM). Inhibition constants (K values) were determined from Dixon plots, as described previously(18) . Kinetic experiments were performed at least twice, and values were averages of at least duplicate experiments. Standard deviations for Kand IC values were <15%.

RNase H Activity Assay

Assays were performed as described elsewhere(15) .

Pyrophosphorolysis Assay

The assay was adapted from the protocol published by Astatke et al.(32) . Pyrophosphorolytic activity is indicated by the cleavage of primer strand at the 3` terminus in the presence of PP(i) and template strand. Briefly, the reaction mixture in 5 µl final volume contained 50 mM Tris-HCl, pH 8.0, 1 mM NaPP(i), 2 mM MgCl(2), 2 nM of the 47/18-mer TP, with the primer P-labeled at the 5` end (1 10^6 disintegrations/min/pmol of 18-mer) and about 250 ng of WT or mutant enzyme. Incubations were for 1 h at 37 °C. The reactions were terminated by the addition of 5 µl of 1% SDS, 40 mM EDTA, 0.06% bromphenol blue, 0.06% xylene cyanol, and 90% formamide. The reaction products were analyzed on a 16% polyacrylamide-8 M urea gel and located by autoradiography(16) .

In Vitro Mutagenesis and Construction of Expression Plasmids

The recombinant clones pET-28a-RT66 and pET-28a-RT51 (16) containing the full-length coding region for p66 and p51 subunits were expressed in Escherichia coli BL-21(DE-3). These vectors also contained the metal binding hexahistidine sequences at the N-terminal region. The XbaI and SacI fragment of pET-28a-RT51 encoding the polymerase domain of RT was subcloned in M13mp18 and used as the template for site-directed mutagenesis(16) . The mutagenesis protocol was essentially the same as described by Kunkel et al.(19) . After ascertaining the mutation in M13, it was introduced into the desired expression cassette and expressed in E. coli BL-21 (DE-3).

Overexpression and Isolation of HIV-1 RT and Its Mutant Derivatives

Cell-free extracts, prepared as suggested by the manufacturer of the pET-28a system (Novagene), were applied to the pre-equilibrated (2.5 ml) Ni-charged IDA-Sepharose column (1 ml/min flow rate). The column was washed extensively until the A was reduced to negligible level. A second wash (5 ml) with a buffer containing 60 mM imidazole, 40 mM Tris, pH 8.0, and 0.5 M NaCl removed all weakly bound proteins. The RT was eluted with 5 ml of 400 mM imidazole, 40 mM Tris, pH 8.0, and 0.2 M NaCl. Active fractions were pooled and dialyzed against 50 mM Tris-HCl, pH 7.0, 1 mM DTT, and 50% glycerol. The eluted proteins were >98% pure, as judged by SDS-polyacrylamide gel electrophoresis(20) . Protein preparations were stable for several months at -20 °C. Different combinations of chimeric heterodimers were prepared by reconstitution of separate subunits, achieved by mixing cell lysates of different bacterial strains at the appropriate ratios(7, 20) . Protein concentrations were determined using the Bio-Rad kit as well as direct spectrophotometric measurements(21) .

Determination of the Rate Constants for First and Second Nucleotide Incorporation by Mutant Enzymes

Rates of incorporation of first ([P]dTMP) and second nucleotide ([P]dAMP) into the 47/18 TP were measured in separate experiments. For the first nucleotide, the standard reaction mixture contained 150 nM TP, 20 µM of [P]dTTP, and 400 nM enzyme. For the second nucleotide, the mixture contained 100 µM non-radioactive dTTP (for complete addition of the first nucleotide) and 20 µM of [P]dATP. Reactions were initiated at 25 °C by the addition of MgCl(2) (2 mM final), and aliquots were withdrawn at the indicated time intervals; incorporation of radiolabeled substrate was determined by acid precipitation. The first-order rate of single nucleotide incorporation in E/template-primer was measured from the slope of the first order plot of ln{[E-TP]/[E-TP]} versus time. The ``initial'' concentration of E-TP was estimated from the amount of dNTP incorporated in E-TP at prolonged incubation (30 min), assuming 1:1 stoichiometry. The amount of E-TP remaining unused at time t is the difference between the amount incorporated at prolonged incubation and the amount incorporated at time t. Incorporation was calculated from the specific radioactivity of [alpha-P]dTTP.

Cross-linking of Enzyme to Template-Primer and Determination of K for DNA

The cross-linking of enzyme to DNA was carried out as described previously(13, 22) , using the 47/18 TP (see Fig. 4for nucleotide sequence). The 5`-end-labeling of the 18-mer was performed using [-P]ATP and T(4) polynucleotide kinase and purified by SDS-polyacrylamide gel electrophoresis(23) . Purification of the primer strand was essential for reproducibility, as the synthetic primers were contaminated with shorter species with lower affinity for the DNA-binding site. The purified primer was appropriately diluted with unlabeled gel-purified 18-mer and annealed to equimolar amounts of unlabeled 47-mer. For the calculation of K for the enzyme-47/18 complex, 60 nM enzyme was incubated on ice for 10 min with variable concentrations of labeled 47/18 (2-100 nM, 100,000 disintegrations/min/pmol). The reaction mixture contained 50 mM HEPES, pH 8.0, 1 mM DTT, 2 mM EDTA, and 5% glycerol. The samples were then exposed to UV light, and the cross-linked species were resolved as before(16) . Dissociation constants (K) were determined by Scatchard plot analysis. In addition, plots of bound DNA versus the logarithm of free DNA were included, in order to ensure that the data actually determine a dissociation constant(24) . Fitting of the data to nonlinear binding curves was performed using the DELTAGRAPH software package.


Figure 4: Qualitative processivity studies on poly(rA)bullet(dT) and 18/47. The two labeled TPs were poly(rA)bullet5`-P-(dT) (A) and 47/5`-P-18-mer (B). Enzymes carrying mutation in either of the subunits were incubated with the labeled TP as described under ``Experimental Procedures.'' Processive reactions were initiated by the addition of dNTP and DNA trap together and incubated at 25 °C for 1 (lane 1) and 10 min (lane 2). Total DNA synthesis (processive and distributive) was initiated by the addition of dNTP alone (no trap) and incubated as above for 1 (lane 3) and 10 min (lane 4). The position of 18-mer primer is shown at the extreme right.



Processivity Studies

1) For qualitative measurements of the processivity of DNA synthesis by mutant and WT enzymes, we used poly(rA)bullet(dT) as well as the 47/18-mer TPs. The 15- and 18-mer primers were 5`-P-labeled and annealed with 2-fold molar excess of the respective template strand. 200-500 nM of enzyme were preincubated with 2 nM of the labeled TP, in an incubation mixture containing 50 mM HEPES, pH 8.0, 80 mM NaCl, 1 mM DTT, 0.1 mg/ml bovine serum albumin, and 2 mM MgCl(2) at a final volume of 3 µl. After a 1-min incubation at room temperature, polymerization was initiated by the addition of 3 µl of 200 µM dNTPs (50 µM each) and 2 mg/ml calf thymus DNA trap(25) . The reaction was allowed to continue at 25 °C; 2.5-µl samples were aliquoted at the indicated time points, mixed with 1 µl of 0.5% SDS and 100 mM EDTA, and frozen in dry-ice until further analysis. The products were analyzed on a denaturing 20% polyacrylamide gel containing 7 M urea, followed by autoradiography.

2) The quantitative processivity studies were essentially as before (26) . Reaction mixtures (final volume 0.5 ml) contained 40 nM WT or 60 nM mutant RTs, 200 nM poly(rA)bullet(dT) (annealed at a 1:1 ratio with respect to the 5`-ends), 0.03 mM [^3H]dNTP, 80 mM NaCl, 1 mM DTT, 50 mM HEPES, pH 8.0, and 0.1 mg/ml bovine serum albumin. MgCl(2) was added to a final concentration of 2 mM to initiate control reactions, MgCl(2) and 100 µM non-substrate homopolymeric trap to initiate the processivity reactions. The mixture was incubated at 25 °C, and aliquots of 25 µl were removed at indicated times and processed as above. We observed no significant effect in the processivity of R72A at dNTP concentrations up to 100 µM.


RESULTS

Overproduction and Purification of Mutant and WT RTs

Our WT RT expression system yielded a protein with kinetic constants similar to other recombinant HIV-1 RTs (Table 1, 18, 16). These results suggest that the hexahistidine regions contained in the recombinant RT did not affect the polymerization mechanism. The level of expressed RT protein, solubility, yield, and the chromatographic characteristics of the mutant and WT enzyme were identical, suggesting that the enzyme structure was intact. In addition, assays performed at different temperatures showed that the activity of WT and mutants dropped concurrently at 55 °C (data not shown), suggesting that all enzymes had the same denaturation temperature, reflecting no changes in the native conformation and three-dimensional folding. Finally, energy calculation of the local effect of the substitutions by the SYBYL molecular modeling program show no major disturbance in the three-dimensional enzyme structure.



Steady State Kinetic Constants

The effect of mutation at Arg of RT was dependent on the TP used to assay the polymerization. The impairment in polymerization was nearly an order of magnitude higher with poly(rA)bullet(dT) than with poly(rC)bullet(dG) (Table 1); the k for poly(rA)bullet(dT) extension decreased by 100-fold for p66/p66 and p66/p51, as compared to p66/p66 and p66/p51, respectively. As the decreases in activity were smaller with poly(dC)bullet(dG), most of the inhibition studies were performed with this TP. There were only minor increases in the K values of the mutant enzymes for dNTP (Table 1). At saturating dGTP concentrations, K for poly(dC)bullet(dG) with all mutant and WT enzymes remained nearly identical, suggesting that there were no significant differences in the affinity of the enzymes for this TP. Comparison of the activities of reconstituted heterodimers to those of homodimers suggests: (i) the activities of homodimer RTs are slightly reduced compared to the corresponding heterodimers, and (ii) the effects of the mutation are through Arg of the p66 subunit, as p66/p51 retains WT activity. In contrast, p66/p51 is similar to p66/p66.

Effect of the Mutation at Arg on the Formation of E-TP

The steady state kinetic experiments suggested that replacement of Arg had no effect on the ability of the enzyme to bind homopolymeric DNA. In order to confirm these results, and to extend them to heteropolymeric sequences, we performed binding studies by direct photochemical cross-linking of the E-TP complex. Irradiation with UV light brings about the formation of a ``Zero length'' covalent bond (27) between the enzyme and the associated TP, thereby, freezing the interaction between them. The DNA binds at the active site of the enzyme in a specific and catalytically relevant manner as demonstrated by the inability of either poly(rA) template or oligo(dT) primer alone to prevent cross-linking, as well as by the competition by poly(rA)bullet(dT) (results not shown). As judged by the extent of cross-linkings, both the mutant and the wild type enzymes show similar binding affinity for the 18/47-mer (Fig. 1). Semilogarithmic graphs of Bound versus Free DNA were fit in binding isotherms [(Bound DNA) = maxBound DNA * Free DNA/K + Free DNA]. At even higher DNA concentrations, the amount of Bound DNA did not increase (results not shown). The dissociation constants calculated from the slopes (-1/K) of Scatchard plots (Fig. 1) were 29 and 25 nM, for the binding of 18/47 to wild type and [R72A] enzymes, respectively.


Figure 1: Binding of template-primer to wild type and R72A HIV-1 RT enzymes. 60 nM of WT (squares) or R72A (circles) were incubated with varying concentrations of 5`-P-18/47-mer (2-100 nM) in a standard reaction mixture (50 µl) and subjected to UV exposure. Samples were processed as described under ``Experimental Procedures.'' The data are plotted in the Scatchard (A) and semilogarithmic graph format (B). K values were calculated from the formula: slope (in graph A) = -1/K. A nonlinear least-squares program was used to fit the data in B to a binding isotherm. [(Bound DNA) = maxBound DNA * Free DNA/K + Free DNA]. Points in the graphs represent the mean values for at least duplicate samples. Standard error was <15%.



Inhibition Studies

A number of residues in the vicinity of Arg seem to be involved in viral resistance to dideoxynucleoside inhibitors, raising the possibility that Arg may be involved in the binding of these compounds. In order to determine if the mutation at Arg affected the inhibition of RT by ddGTP, a typical dose-response study was carried out. As shown in Table 2, the IC of ddGTP inhibition with poly(dC)bullet(dG) as TP by WT and R72A are unaffected. Nevirapine, known to bind at a different site of the RT polymerase domain(5) , was also equally effective in inhibiting the mutant and WT enzymes (Table 2). These results show that Arg does not participate in the binding of these inhibitors. However, R72A exhibited moderate susceptibility to inhibition by PP(i). The inhibition constants (K) calculated from Dixon plots (not shown) were 0.71 and 0.58 mM for R72A and WT, respectively, with poly(dC)bullet(dG) as the TP (Table 2). Most interestingly, PFA, a PP(i) analog, did not inhibit R72A even at millimolar concentrations (Table 2). Resistance of R72A to PFA was also observed when 18/47 was used as TP. It may be pointed out that the K for the inhibition of WT RT by PP(i) or PFA are significantly lower with poly(rA)bullet(dT) as TP (10, 18) ; however, since R72A does not utilize this TP efficiently, we could not ascertain insensitivity of poly(rA)bullet(dT)-directed DNA synthesis to PFA. The effects of the mutation on the susceptibility of RT to PFA are through Arg of the p66 subunit, as p66/p51 retains WT kinetic constants and inhibition profile (Table 2). In contrast, p66/p51 is not inhibited by PFA (Table 2). These results suggest that Arg of p66 may be located at (or proximal to) the PP(i)-binding site and/or there are subtle, yet potentially substantial differences in the binding of PP(i) and PFA to RT that are magnified in the absence of arginine at the 72 position. Interestingly, R72K retained sensitivity to PFA (results not shown).



Pyrophosphorolysis Assay

In order to verify that the defect of R72A is in the pyrophosphate binding/release function, we examined the ability of WT and mutants to perform the reverse reaction, namely pyrophosphorolysis. As seen in Fig. 2, the WT RT utilizes PP(i) to hydrolyze the 18-mer primer of the 47/18 TP, as noted by the appearance of smaller size DNA fragments (lane 2). No pyrophosphorolysis products are observed in the absence of PP(i) (Fig. 2, lanes 1, 3, and 5). Interestingly, R72A with mutations in only p66 (Fig. 2, lanes 3and 4) or in both subunits (Fig. 2, lanes 5 and 6) was unable to catalyze pyrophosphorolysis (Fig. 2, lanes 4 and 6), presumably, due to inability of R72A to bind PP(i) in the absence of Arg.


Figure 2: Pyrophosphorolysis reaction by wild type and R72A. Reactions were performed as described under ``Experimental Procedures'' for the pyrophosphorolysis of P-labeled 47/18-mer TP (1 10^6 disintegrations/min/pmol of 18-mer) in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of 1 mM PP(i). WT is shown in lanes 1 and 2, p66/p51 in lanes 3 and 4, and p66/p51 in lanes 5 and 6. Lane 7 contains only P-labeled 18/47-mer.



Processivity of Mutant Versus WT RT

Another assessment of the polymerase function is its ability, once bound to TP, to successively add dNTPs to the growing chain prior to dissociation from the replication complex. When either WT RT or R72A were incubated with saturating amounts of TP, the polymerization reaction time course followed the following relationship N/E = (k/k)(1 - exp(-kt)). N/E is the number of additions/processive cycle/mole of E-polymer, k is the steady state rate constant for polymerization, and k is the rate constant for the dissociation of E-polymer(20) . After a sufficiently long time period, (t1/k), a true plateau representing the complete dissociation of all productively bound RT molecules was achieved. The plateau is described by the relationship (N/E) = k/k. (N/E) is the limiting value for incorporation of dTTP into polymer. Values for k were calculated from control reactions where the DNA trap was omitted from the reaction protocol(27) . The results of two trap experiments are summarized in Fig. 3. These data indicate that the majority of p66/p51 molecules are able to extend the primer only by one nucleotide in the presence of trap. Additional evidence to support the defect of mutant in processivity was obtained using poly(rA)(dT) as TP (Fig. 4). In this experiment, the enzyme was first incubated with poly(rA)P(dT), followed by the addition of both trap and TTP. The extension of P-labeled (dT) was then monitored at 1 or 10 min time intervals, and the products were analyzed on a 20% urea-polyacrylamide gel (Fig. 4A, lanes 1 and 2 of p66/p51). Most interestingly, the mutant enzyme even in the absence of DNA trap extended the poly(rA)(dT) TP by only one or two nucleotides (Fig. 4A, lanes 3 and 4 of p66/p51). In contrast, WT was efficient in RNA-directed DNA synthesis, with or without DNA trap (Fig. 4A, lanes 1-4 of p66/p51).


Figure 3: Processivity studies on poly(rA)bullet(dT) for wild type and R72A RTs. Reactions were performed as described under ``Experimental Procedures'' for incorporation of TMP into poly(rA)bullet(dT), by WT (A) and R72A (B). The curves represent the best fit of the data N/E = (k/k)(1 - exp(-kt)). N/E is the number of nucleotides added per processive cycle by an enzyme molecule (y axis)(27) . The amount of background incorporation with DNA trap preincubation was <8% of the respective activities in unchallenged experiments (filled triangles). The corrected values for the processive conditions in the presence of trap are presented in the graphs (open squares).



Similar, though less dramatic, results were observed with 47/18-mer as TP. In the presence of trap it is observed that the majority of extended primers by p66/p51 in 1 or 10 min are of the n+1 size (n = size of original primer) (Fig. 4B, lanes 1 and 2 of p66/p51). However, without DNA trap, the same enzyme is able to add a few more nucleotides (Fig. 4B, lanes 3 and 4 of p66/p51). Once again, a substantial number of WT RT molecules (Fig. 4B, lanes 1-3 of p66/p51) are able to copy the full-length of template in the presence or absence of trap.

The reduced processivity of R72A probably resulted from the extended stalling of p66/p51 by remaining bound to the TP, followed by dissociation from the TP. Evidence for the former is the observed increase of the product size with time under processive conditions (increasingly higher intensity of lower bands in Fig. 4A, lanes 1versus2, for p66/p51). Under these conditions (trap present), extension of product can only be done by the same enzyme molecule, as the trap binds effectively all free and dissociating enzyme molecules (the effectiveness of trap to arrest free enzyme is shown in Fig. 4(extreme right lane), where no polymerization by WT was observed in 10 min, when the trap was added at the binding step). The effects of the Arg mutation are exerted through the p66 subunit, as judged by the processivity profiles of p66/p51 and p66/p51. The mutation in p51 affects neither the activity nor the processivity, whereas mutation in p66 alone, decreases both processivity and polymerization activity.

Rates of First and Second Nucleotide Incorporation by WT and R72A

In order to ascertain the contention that the mutation at Arg severely disables the enzyme from polymerizing processively, we measured the rates of incorporation of first and second nucleotide into the 47/18-mer TP containing dA and dT as first and second available template base, respectively. If translocation is slower than polymerization or, alternatively, if the enzyme falls off from the template strand after the first addition, then the incorporation of the first nucleotide [P]TTP (in the absence of other nucleotides) will be faster than that of the second nucleotide [P]dATP (in the presence of excess nonradioactive TTP). As shown in Fig. 5, although the first nucleotide is added at a modest 2-3-fold lower rate for R72A compared to WT, the second nucleotide is added by the mutant 45 times more slowly, as judged by the ratio of slopes in Fig. 5, A and B. These results are in agreement with the processivity analysis, suggesting that the mutation introduces an impairment that results in the inability of the enzyme to polymerize past the first nucleotide. Furthermore, the ability of RT to catalyze a single addition without having to translocate is only moderately affected.


Figure 5: Rates of first and second nucleotide incorporation by wild type and R72A RT. The rate of incorporation of first nucleotide (A) was measured by monitoring the incorporation of [alpha-P]TMP into 18/47 (see Fig. 4for nucleotide sequence) in a reaction mixture containing 50 mM HEPES, pH. 7.5, 2 mM MgCl(2), 400 nM enzyme, and 20 µM [alpha-P]TTP (1.4 µCi/nmol). Reactions were carried at 25 °C and aliquots were removed at indicated times and the amount of [P]TMP incorporated was determined as described under ``Experimental Procedures.'' The rate of second nucleotide (dATP) addition (B) was measured in a similar manner, except that 100 µM of unlabeled first nucleotide (TTP) was also added along with 20 µM of [alpha-P]dATP (1.4 µCi/nmol). Open squares and filled circles represent values for WT and R72A, respectively. The first-order rate of single nucleotide incorporation in E/template-primer was measured from the slope of the first-order plot of ln{[RT-TP]/[RT-TP]} versus time (slope= -k/2.3). [RT-TP] is estimated from the amount of dNTP incorporated in E-TP at prolonged incubation (30 min), assuming 1:1 stoichiometry. [RT-TP] is estimated from the difference between [RT-TP] and the amount incorporated at time t.




DISCUSSION

The characterization of the available large number of HIV-1 RT mutants (9, 28, 29, 38) has not been extensive with regard to the polymerization mechanism. Hence, the role of the mutated residues in the catalytic mechanism of RT has remained elusive. Furthermore, with the exception of a few studies(7, 30) , the vast majority of mutagenesis studies have not been subunit specific, leaving an uncertainty regarding the specific residue responsible for the observed effect. We have shown in our laboratory (12) that 10 residues of KF of E. coli DNA polymerase I have equivalent counterparts in the three-dimensional structure of HIV-1 RT, suggesting functional equivalence of these residues. In this study, Lys of the KF was reported to be spatially equivalent to Arg of RT. However, with the availability of the new crystal structure coordinates for KF(31) , we now find that Arg is the equivalent residue to Arg. (^2)Nevertheless, the catalytic roles of both Arg or Lys, as judged by site-directed mutagenesis appear similar(32) . Furthermore, Lys has been extensively characterized through chemical modification and site-directed mutagenesis studies(33, 13) . The mutational studies suggested its involvement in (a) dNTP binding and (b) translocation along the template strand(13) . In addition, the recent crystallographic studies of KF complexed with PP(i) and dNTP (31) have implicated Lys as a probable binding site for the beta- phosphates (PP(i)) of dNTP. With regard to Arg of KF, its importance in the catalytic function is implied by a significant loss of activity of R754A(32) . Furthermore, it was also suggested to play a major role in PP(i) binding (32) . Since in RT, Arg is now proposed to be a counterpart of Arg, we examined the role of Arg in the catalysis of DNA synthesis by RT. We constructed and characterized RTs with conservative and non-conservative mutations at Arg. Incidentally, a conservative R72K mutant had been constructed previously and moderate reduction in the activity of cell extracts containing the mutant enzyme was reported, without further investigation of the role of this residue (9) .

We found Arg to be required for efficient polymerization. Interestingly, the catalytic properties of Arg could be partially compensated by lysine, as judged by the small reduction in the k of R72K (Table 1). These results suggest that the participation of Arg in the catalytic process is of electrostatic nature. Nevertheless, the significant loss of polymerase activity associated with the R72A mutation could not be accounted for by the minor changes in the K for dNTP. We therefore examined if the R72A has reduced DNA binding ability. The x-ray structure of RT complexed with double-stranded DNA had suggested a role for the Arg containing region in the binding of DNA(6) . Our examination clearly showed that Arg does not participate in DNA binding, as the K and K for DNA TP of R72A, calculated from kinetic and DNA cross-linking experiments (Table 1) were comparable to those of the WT enzyme. Thus, the defect in the polymerase reaction catalyzed by the mutant enzyme and presumably the functional role of Arg appeared to lie at a step beyond the EbulletTP complex formation. We therefore considered a possible shift from processive to distributive mode of DNA synthesis by R72A. Our experiments ( Fig. 3and Fig. 4), clearly show that the p66/p51 enzyme lost its ability to polymerize in the processive mode (Fig. 4). Furthermore, as expected Arg of only the p66 subunit is necessary for processive polymerization. Additional insight into the catalytic defect of R72A was provided by kinetic experiments. First-order kinetics (Fig. 5) showed that the first nucleotide is added by the mutant and WT enzymes at comparable rates (Fig. 5). However, the rate of subsequent nucleotide addition was significantly reduced only in the case of R72A. Possible affected steps between the addition of first and second nucleotides are either the removal of pyrophosphate or the translocation along the template strand. Our experimental evidence argues for involvement of Arg in the former step, as R72A exhibits a total resistance to PFA, a PP(i) analog (see below). Furthermore, our pyrophosphorolysis results strongly support the involvement of Arg in the pyrophosphate binding function, as in the absence of Arg the mutant is severely impaired in its ability to catalyze pyrophosphorolysis of DNA (Fig. 2). In addition, computer-assisted modeling of a prepolymerization complex consisting of HIV-1 RT, TP, and Mg-dNTP (16) shows that the Arg side chain is oriented toward the PP(i) moiety of dNTP. Finally, the crystal structures of KF with PP(i) or dNTPs show the side chains of Arg as well as Lys (the equivalent of Arg residue in the RT enzyme) providing the PP(i)-binding site(31) . A recent report on the properties of R754A has also suggested a role for R754 in PP(i) binding(32) . Therefore, the primary role for Arg in catalysis appears to be related to binding and/or removal of PP(i). A plausible consequence of such a defect could very well be seen in the inability of R72A to translocate across the template; however, the possibility of participation of Arg in the PP(i)-binding-independent translocation event cannot be excluded.

In our drug susceptibility studies, R72A and WT RTs were found to be equally sensitive to nucleotide analog as well as nevirapine, implying non-involvement of Arg in the binding of these inhibitors. In regard to PP(i) inhibition, a small but consistent increase in the K for PP(i) was noted with R72A (Table 2). However, the most prominent effect of the R72A mutation was seen in the form of total resistance of mutant enzyme to PFA (Table 2). Despite the similarity in the structures of PP(i) and PFA, we find R72A to discriminate among these two inhibitors. The differences in the inhibitory effects may be attributed to the different size and charge of the two molecules, which presumably causes the large difference in their K values, even for the WT enzyme (Table 2, and (18) ).

In addition to Arg, other RT residues (Glu, Asp, Ala, and Tyr) (34, 35) may also be involved in the mechanism of resistance to PFA. From these mutants, GluGly and TyrAsn have been shown to be almost totally resistant to PFA, similar to ArgAla reported in this communication. As these three residues (Glu, Arg, and Tyr) are significantly apart in the three-dimensional structure(5, 6) , it is unlikely that they directly interact with PFA at a common binding site. The following reasoning shows that only Arg could participate directly in the PFA/PP(i) binding: as the Tyr interacts through its phenyl ring in a hydrophobic manner (TyrPhe has WT properties)(36) , it seems unlikely for this residue to directly interact with the polar PFA molecule. In addition, our modeling studies show the Tyr side chain poised to interact with the ribose ring of the dNTP which is significantly away from the pyrophosphate moiety of dNTP. Similarly, the negative charge of Glu makes any direct interaction with PFA unlikely. The fact that ArgLys retains sensitivity to PFA, suggests that the RT-PFA interaction is based on electropositive charge at the 72 position. While examining the metal preferences of WT and mutant enzymes, we observed a 2-3-fold increase in the Mn/Mg activity ratio (results not shown). This change in metal specificity is consistent with Arg interacting with the metal-binding beta- phosphate moiety of the dNTP.

In conclusion, we have shown that Arg is a catalytically important residue for RT, required for PP(i) binding, release, and the translocation function. To our knowledge, this is the first residue of HIV-1 RT, outside the thumb subdomain(37) , experimentally shown to contribute to the processive mode of polymerization. It therefore appears that the ability for processive polymerization is not exclusively due to residues of the thumb subdomain, but to other amino acids, as well.


FOOTNOTES

*
This work was supported in part by Grant 26652 from the National Institute of Allergy and Infectious Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 201-982-5515; Fax: 201-982-5594.

(^1)
The abbreviations used are: RT, reverse transcriptase; dNTP, deoxynucleotide triphosphate; poly(dC)bullet(dG), polydeoxycytidine-(deoxyguanosine); foscarnet or PFA, phosphonoformic acid; KF, Klenow fragment; PP(i), pyrophosphate; poly(rA)bullet(dT), polyriboadenosine-(thymidine); poly(rC)bullet(dG), polyribocytidine (deoxyguanosine); TP, template-primer; WT, wild type; DTT, dithiothreitol. Kinetic nomenclature and constants are according to Cleland (1). Unless indicated otherwise, the terms R72A and R72K refer to p66/p66 and p66/p66 respectively.

(^2)
S. G. Sarafianos, V. N. Pandey, N. Kaushik, and M. J. Modak, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Satish Sharma of the Upjohn Company, for providing the DE52 plasmid containing the HIV-1 RT gene, Dr. S. Wilson for providing the pRC-RT construct expressing HIV-1 RT, and Richard Whipple for help with the cross-linking technique at the early stages of the work.


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