Kinetic Analysis of Four HIV-1 Reverse Transcriptase Enzymes Mutated in the Primer Grip Region of p66

The highly conserved primer grip region in the p66 subunit of HIV-1 reverse transcriptase (RT) is formed by the β12-β13 hairpin (residues 227–235). It has been proposed to play a role in aligning the 3′-OH end of the primer in a position for nucleophilic attack on an incoming dNTP. To analyze the importance of the primer grip for RT function, mutant RTs were used that contain single alanine substitutions of residues Trp229, Met230, Gly231, and Tyr232 in the p66 subunit of the heterodimeric p66/51 enzyme. Steady-state and pre-steady-state kinetic analyses of the enzymes were performed. All mutant enzymes revealed reduced polymerase activity. Mutation of Y232A showed the smallest effect on polymerase function. Equilibrium fluorescence titrations demonstrated that the affinity of the mutants for tRNA was only slightly affected. However, the affinity for primer-template DNA was reduced 27-fold for mutant p66W229A/51 and 23-fold for mutant p66G231A/51, and the maximal pre-steady-state rate of nucleotide incorporation,k pol, was reduced 27-fold for p66W229A/51 and 70-fold for p66G231A/51, respectively. Mutant p66M230A/51 revealed no reduced affinity for primer-template but showed a 71-fold reduced affinity for dTTP. Additionally, the mutations Trp229 and Gly231 affected the stability of the RT heterodimer.

Reverse transcriptase (RT) 1 is pivotal to the replication of retroviruses. The RT of human immunodeficiency virus 1 (HIV-1) is a heterodimeric enzyme composed of a p66 and a p51 subunit that share a common amino terminus. The smaller subunit is a cleavage product of p66 and lacks the RNase H domain (1,2). RT is a multifunctional enzyme that possesses a DNA-dependent and an RNA-dependent DNA polymerase activity and an RNase H activity that cleaves RNA in RNA/DNA hybrids. In addition to these activities it uses a host-derived tRNA 3 Lys molecule as a primer for the first round of polymerization. Furthermore, it has been shown that HIV RT is only active as a dimeric enzyme and that the monomeric subunits show no catalytic activities (3,4).
For HIV-1 RT and for other DNA polymerases, a polymerization mechanism has been proposed where the binding of primer-template precedes the binding and incorporation of the first dNTP (5). A model for the mechanism of RT polymerization is shown in Scheme 1. In this model, the first step of the reaction is thought to be the formation of a collision complex of RT and DNA that is concentration-dependent. This is succeeded by a conformational change to form a tight complex, which in turn is a prerequisite for dNTP binding. A second conformational change is necessary after the initial binding of the dNTP to form a tight ternary complex. This was identified as the rate-limiting step prior to nucleotide incorporation (6). RT* ⅐ p n /t RT* ⅐ p n /t ϩ dNTP K 3 % RT* ⅐ p n /t ⅐ dNTP K 4 *RT* ⅐ p n /t ⅐ dNTP K 5 % *RT* ⅐ p nϩ1 /t ϩ PP i SCHEME 1. Model for the kinetic mechanism of polymerization by RT. The asterisks indicate different conformational states of the RT.
One of the highly conserved motifs in retroviral RTs spans the region from residue 220 to 243 (7). Part of this region in p66 constitutes the "primer grip" and forms a hairpin structure that ranges from residue 227 to 235 (␤12-␤13 hairpin, adopting the nomenclature of Ref. 8). The corresponding residues in the p51 subunit are unraveled in the crystal structure (9). In the RT/DNA cocrystal the primer grip is located in close vicinity to the polymerase active site and was therefore proposed to be important for maintaining the 3Ј-OH end of the primer in the appropriate orientation for nucleophilic attack on an incoming dNTP (8,9). Especially the amino acids in the loop connecting ␤12 and ␤13 (Met 230 and Gly 231 ) and the residues adjacent to the loop, namely Trp 229 at the end of ␤12 and Tyr 232 at the beginning of ␤13, are close to the nucleic acid and could play a role in orienting the DNA primer. The analysis of regions that are necessary for RT function may contribute to the design of more efficient inhibitors targeting essential residues.
To investigate the importance of the primer grip region for RT function, mutant RTs were used that contain single alanine * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  substitutions of residues Trp 229 , Met 230 , Gly 231 , and Tyr 232 only in p66. The mutant p66 polypeptides were reconstituted into heterodimer in vitro with a wild type p51 subunit (10 -12). Pre-steady-state and steady-state kinetic methods were used to analyze tRNA and primer-template binding, as well as nucleotide binding and incorporation of the mutant enzymes.

EXPERIMENTAL PROCEDURES
Proteins-Selectively mutated p66/51 HIV-1 RT and WT RT were prepared by in vitro reconstitution as described previously (10,11,13). Briefly, biomass from Escherichia coli cultures expressing the mutant p66 polypeptide was mixed with that of a strain expressing His 6 -p51 and co-homogenized. The high speed supernatant was loaded on a Ni 2ϩ -nitrilotriacetic acid-Sepharose (Qiagen) column and eluted with a 0 -0.5 M imidazole gradient. RT-containing fractions were pooled, dialyzed, and applied on a DEAE-Sepharose column. The flow-through of the column was then applied onto a HiTrap SP column (Pharmacia Biotech Inc.) to separate the dimeric fraction from excess His 6 -p51 and eluted with a 0 -0.5 M NaCl gradient. Samples were dialyzed against a buffer containing 50% glycerol and stored at Ϫ20°C. The purified RTs were free of nuclease contamination.
HPLC Gel Filtration Analysis-HPLC gel filtration analysis was performed using a Superdex 200 HR10/30 column. 5-30 g of enzyme were loaded onto the column. The column was eluted with HPLC buffer at 0.5 ml/min. tRNAs-In vitro transcription and purification of tRNA 3 Lys was performed as described previously (14). E. coli tRNA 2 Trp was purchased from Sigma.
Fluorescence Equilibrium Titrations with tRNA-Fluorescence titrations were performed using an SLM Smart 8100 spectrofluorometer equipped with a PH-PC 9635 photomultiplier. Intrinsic fluorescence measurements using the tryptophan fluorescence of the enzymes were performed in reaction buffer in a total volume of 0.7 ml or 1 ml as described previously at an excitation wave length of 295 nm. The emission intensity was measured at 340 nm (slit widths of 1 and 16 nm for excitation and emission, respectively) (14). Values for the dissociation constant (K d ) were determined as described previously (15) using the data-fitting program Grafit (Erithacus Software). A quadratic equation analogous to the one given by Mü ller et al. (15) was used for the fitting procedure.
Oligodeoxynucleotides-Oligodeoxynucleotides were synthesized on an Applied Biosystems 380 B DNA synthesizer and purified by HPLC on a reversed phase column by standard procedures (15) or by denaturing polyacrylamide gel electrophoresis (10% acrylamide, 7 M urea) and elution of the desired DNA band. The sequence of the 18-mer primer oligonucleotide was 5Ј-TCCCTGTTCGGGCGCCAC, and that of the corresponding 36-mer template oligonucleotide was 5Ј-TGTGGAAAATCT-CATGCAGTGGCGCCCGAACAGGGA. The complementary regions are equivalent to the primer binding site sequence from HIV-1. The sequence of the fluorescently labeled primer oligodeoxynucleotide was identical to the one above with the fluorescent label at the 5Ј-end. It was synthesized by coupling the phosphoramidite dye 6-FAM, a fluorescein derivative, directly to the 5Ј-end of the oligodeoxynucleotide during DNA synthesis according to the recommendations of the manufacturer (Applied Biosystems). For the fluorescently labeled primer oligonucleotide, 5Ј-TGTGGAAAATCTCTAGATGTGGCGCCCGAACAGGGA was used as a template. Primer and template oligonucleotides were annealed by heating equimolar amounts in annealing buffer for 2 min at 90°C, followed by cooling to room temperature over 1 h in a water bath.
Fluorescence Equilibrium Titrations with Primer-Template-Experiments with the 5Ј-fluorescently labeled FAM-primer-template (p/t) were performed and evaluated as described above. The excitation was at 492 nm, and the emission intensity was measured at 516 nm (slit widths set at 1 and 8 nm for excitation and emission, respectively). For displacement titrations, a preformed complex of 30 nM RT and 30 nM of fluorescently labeled p/t DNA was titrated with unlabeled p/t in reaction buffer. The increase in fluorescence was used as a signal for displacement of labeled p/t. Data were analyzed by using the fitting program Scientist to determine the K d values for the unlabeled p/t.
5Ј-End Labeling of Primers-Primer oligodeoxynucleotides were 5Јend-labeled with T4 polynucleotide kinase (New England Biolabs) using 7 Ci of [␥-32 P]ATP (3000 Ci/mmol) (Hartmann Analytic, Braunschweig) per 100 pmol of DNA for 1 h at 37°C. The primer was separated from nucleotides by purification over a NucTrap Probe Purification Column (Stratagene) according to the procedure described by the manufacturer. Concentrations were determined by thin layer chromatography using a phosphor imaging device (Bio-Rad) for quantification.
Rapid Kinetics of Nucleotide Incorporation-Rapid quench experiments were carried out in a rapid quench apparatus built by KinTek Instruments (University Park, PA) in reaction buffer. 15 l of a preformed complex of 100 nM RT or higher concentrations as indicated for the mutants in the figures and p/t DNA (200 nM) were rapidly mixed with 15 l of dTTP (up to concentrations of 5 mM). Reactions were quenched at various time points with 0.6% trifluoroacetic acid. The products were analyzed on denaturing 10% polyacrylamide gels with 7 M urea. Quantification of the data was achieved by phosphor imaging. All concentrations reported are final concentrations after mixing in the rapid quench apparatus. Data were fitted to a burst equation (single exponential plus slope) using the program Grafit. The pre-steady-state rate is given by the exponential (6). The burst amplitude corresponds to the amount of RT bound to p/t at the beginning of the reaction (t ϭ 0).
Affinities for dTTP were determined by the dependence of the presteady-state burst rate on the dTTP concentration. The RT-p/t complex (200 nM p/t with RT as indicated in Fig. 5 for the various enzymes) was rapidly mixed with increasing concentrations of nucleotide and quenched after t1 ⁄2 of the maximal pre-steady-state rate. The corresponding rates were calculated from the elongated fraction of the primer by converting the single exponential equation into k ϭ Ϫln((1 Ϫ fraction of elongated primer/burst amplitude))/t1 ⁄2 . The observed rates were then plotted against the nucleotide concentration, and the K d for dTTP was calculated by fitting the data to a hyperbola using the program Grafit.
Primer Extension Assays-primer extension assays were performed with 200 nM radioactively labeled p/t and 100 or 450 nM dimeric RT enzyme at 25°C in reaction buffer in a total reaction volume of 10 l. Reactions were started by adding dNTPs as indicated in Fig. 6 and were stopped after 5 min by the addition of 10 l of urea-loading buffer.

Effect of Mutations on RT Polymerase
Activity-To obtain a first impression of whether the introduction of mutations into the primer grip region has an impact on RT function, we assayed the RNA-dependent DNA-polymerase activity of the reconstituted wild type and mutant enzymes on the homopolymeric substrate poly(rA)/oligo(dT) [12][13][14][15][16][17][18] . Table I shows that the RT activity of all mutants is reduced. The most severe reduction of RT polymerase activity can be detected with p66 W229A / 51. Previous primer extension experiments with a synthetic 36/71-mer p/t DNA had already indicated that this mutant is strongly impaired in polymerase activity (11). However, no detailed kinetic analyses of the polymerase function of the mutants were available.
The reduction of RT activity of the mutants may derive from different mechanisms. First, the mutation can have an effect on  (3), it is important to determine the dimer content of the purified enzymes, especially since the enzymes used were reconstituted from subunits expressed separately.
N-terminal tagging of the p51 subunit with six additional histidines allows purification by metal chelate chromatography. The wild type or mutated p66 subunit, which in our case does not have a histidine extension, can only be retained on the column if it interacts with p51 (10). Although metal chelate chromatography (16) of the in vitro reconstituted HIV-1 RT mutants is a powerful method that permits purification of the dimer fraction of an enzyme, it does not give much information on the stability of the purified dimer. If the dimer is not stable, a dimer/monomer equilibrium will be reestablished after purification of the dimeric enzyme fraction. To investigate this possibility, we determined the dimer content of the RT enzymes by HPLC size exclusion gel filtration ( Fig. 1) after the proteins had been purified and dialyzed against storage buffer. The results, shown in Table II, indicate that the mutants p66 W229A /51 and p66 G231A /51 are not completely dimerized. p66 W229A /51 is only about 35% dimeric, and the dimer content of p66 G231A /51 is about 60%. Therefore, the low polymerase activity of mutants p66 W229A /51 and p66 G231A /51 is partly due to the low dimer content of the purified stock solutions. This result indicates that the dimer, due to its instability, tends to monomerize during the purification procedure. To account for this result, for all the analyses shown below only the dimer content of the enzymes was considered for calculations. The dimer interface of the mutants p66 M230A /51 and p66 Y232A /51 appears to be intact, since we could not detect appreciable amounts of monomer.
To examine whether the enzymes p66 W229A /51 and p66 G231A /51 are stable under the conditions used in our kinetic experiments (25°C for up to 1.0 h) we isolated the dimer fraction of mutants p66 W229A /51 and p66 G231A /51 by separation on an HPLC gel filtration column. This is shown in Fig. 1, C and D, for p66 W229A /51. Immediately after collection, an aliquot of the collected dimer fraction was loaded again on the HPLC gel filtration column to show the dimer content of the isolated peak fraction (Fig. 1C). Another aliquot was incubated for 1.0 h at 25°C and then analyzed again by HPLC gel filtration (Fig.  1D). The mutant enzymes remained dimeric for up to 4.5 h of incubation at 25°C (data not shown). This suggests that the proteins do not dissociate to a great extent under the conditions in which our experiments were performed. However, the wild type and mutant enzymes started to dissociate appreciably after 15 min at 37°C, a temperature that in general is used for many enzyme assays with RT (data not shown).
tRNA Binding Is Not Impaired-tRNA 3 Lys is the natural replication primer of HIV-RT. We have shown recently that the intrinsic fluorescence of RT can be used to monitor p/t and tRNA binding (14,17). Heterodimeric RT possesses 37 tryptophan residues. Binding of tRNA or p/t to RT leads to a quenching of the tryptophan fluorescence of the protein that can be used to determine the dissociation constant (K d ). However, comparison of binding affinities for WT RT to various tRNA species showed that RT does not bind tRNA 3 Lys specifically; instead, for all tRNA species tested, similar K d values were determined (14). Therefore, to determine whether the mutant RTs are still able to bind tRNA we used E. coli tRNA 2 Tyr for the intrinsic fluorescence titrations (Fig. 2). The results presented in Table III indicate that mutations in the primer grip do not affect tRNA binding. This was confirmed by titration of wild type and mutant RT p66 W229A /51 with synthetic tRNA 3 Lys . The K d values obtained for synthetic tRNA 3 Lys were 11 nM for the wild type and 7 nM for p66 W229A /51. Our results demonstrate that the primer grip region is not essential for tRNA binding.
Influence on Primer-Template Binding-The affinity of the mutant RTs for p/t DNA was determined by equilibrium fluorescence titrations using a 18/36-mer p/t DNA substrate labeled at the 5Ј-end of the primer with the fluorescein derivative FAM. The K d values obtained for the wild type enzyme with this method are similar to those obtained by intrinsic fluorescence measurements (K d ϭ 1.8 nM; Ref. 17). Since some of the mutants have rather high K d values for p/t, for practical reasons we used the extrinsic fluorescence determination for standard procedures because the signal change is larger and more stable (Fig. 3). The binding affinities for p66 W229A /51 and p66 G231A /51 were about 23-27-fold lower than for wild type, whereas the binding affinity for mutant p66 Y232A /51, which possessed the highest RT polymerase activity (Table I), was only about 8-fold lower. Interestingly, the mutation of p66 M230A /51 does not appear to have an effect on p/t binding. This result indicates that this mutant, which retains only 11% of the WT RT polymerase activity must be impaired in one of the steps following p/t binding. However, to exclude the possibility that the fluorescence label has an influence on the affinity of the mutant for p/t, we performed displacement titrations. A preformed complex between RT and the fluorescent p/t was titrated with the unlabeled 18/36-mer DNA. Displacement of the labeled p/t is dependent on the K d of both p/t DNAs. Analysis of the displacement data for the wild type and p66 M230A /51 leads to K d values for the unlabeled p/t species of 5.2 and 5.3 nM, respectively, which indicates that the label does not influence the binding affinities of the mutant and confirms that the affinity of wild type and p66 M230A /51 for p/t is unchanged.

Kinetics of Nucleotide Incorporation
Are Changed-To analyze the kinetics of nucleotide binding and incorporation we used quench flow methods (6). Elongation of the 18/36-mer DNA to the 19/36-mer product was achieved by rapid mixing of the preformed enzyme-DNA complex with various concentrations of the next correct deoxynucleotide dTTP in a quench flow apparatus. Fig. 4 shows a typical time dependence of dTTP incorporation at a concentration of 1 mM. All enzymes incorporated dTTP rapidly in a burst of polymerization followed by a slower steady state reaction (slope of the fitted curves), which is dependent on the off-rate of p/t binding. The burst amplitude corresponds to the amount of RT bound to p/t at the beginning of the reaction. Both the burst amplitude and the burst rate are, among other things, dependent on the affinity of the enzyme for p/t. Mutants having high K d values for p/t DNA (p66 W229A /51, p66 G231A /51, and p66 Y232A /51) showed smaller burst amplitudes and reduced burst rates. For p66 W229A /51 and p66 G231A /51, the enzyme concentration had to be increased to detect the burst (Fig. 4B). To reach the maximal pre-steadystate rate of nucleotide incorporation (maximal burst rate, k pol ), elevated dTTP concentrations of up to 5 mM had to be used for mutants p66 W229A /51, p66 M230A /51, and p66 G231A /51, indicating a lower affinity for nucleotides (see below). With WT RT, the maximal rate is already achieved at a dTTP concentration of 150 M. The k pol values obtained for dTTP incorporation for all enzymes are shown in Table IV.
The method described here for the determination of k pol can also be applied to determine the apparent affinities of dTTP to the RT-DNA complex by measuring the dependence of the pre-steady-state burst rate on the concentration of dTTP (6). The rates increase with increasing dTTP concentrations used.  Table III.  The observed rates at different dTTP concentrations were plotted against the dTTP concentration, and the dissociation constant (K d ) for dTTP was calculated by fitting the data to a hyperbola (6) (Fig. 5). Again, mutant p66 Y232A /51 was the least impaired. In addition to their low affinity for p/t DNA, the affinity of mutants p66 W229A /51 and p66 G231A /51 for dTTP was reduced 20-and 36-fold, respectively. Our data clearly show that the major effect of the mutation in p66 M230A /51 is a severe increase (71-fold) in the K d for dTTP. A summary of the results is given in Table IV.
Polymerization on a Heteropolymeric DNA Substrate- Fig. 6 demonstrates that the results of the equilibrium titration and single turnover experiments can be confirmed in a polymerization assay. A primer extension assay was performed using the same radioactively labeled p/t (200 nM) as substrate as applied in the quench flow experiments and increasing amounts of dNTPs and RT. In this experiment, p66 Y232A /51 was only slightly less active than the wild type enzyme even when low dNTP concentrations of 50 M were used. p66 M230A /51 needs higher dNTP concentrations to perform efficient polymerization to the end of the template. With mutants p66 W229A /51 and p66 G231A /51, as expected from the results shown above, both an increase in the enzyme and dNTP concentrations is necessary for proficient polymerization. DISCUSSION The aim of this work was to characterize the importance of the primer grip of HIV-1 RT by kinetic analyses of four mutant RT enzymes. Comparison of the primer grip region of HIV-1 RT with RTs from other retroviruses possessing a p66/51 structure (feline immunodeficiency virus, simian immunodeficiency virus, equine infectious anemia virus, and HIV-2) shows that Pro 225 , Pro 226 , Trp 229 , Gly 231 , and Leu 234 are invariant, indicating an important role of these residues for enzyme function (7). Residues Trp 229 and Leu 234 of HIV-1 p66 also participate in the binding of non-nucleoside inhibitors (NNIs). Although in the cocrystal structure Trp 229 and Leu 234 are in close contact with the inhibitor nevirapine (Յ3.8 Å), no mutant resistant to NNIs has been found with changes in these amino acid residues (18 -21). Targeting of residues in the NNI binding site that are essential for RT function could be important for the design of novel inhibitors that do not lead to resistant virus easily.
Previous biochemical analyses with p66 W229A /51 already showed that this enzyme is strongly impaired in polymerase function (11). The instability of the dimer we observed with p66 W229A /51 and p66 G231A /51 is somewhat surprising, since the amino acids constituting the primer grip are not directly involved in dimer formation. However, it has been demonstrated that a p66 subunit with a mutation of Leu 234 to Ala completely  Table IV.  5. Dependence of the pre-steady-state burst rate on dTTP concentration. Increasing amounts of dTTP rapidly mixed to a preincubated solution of 200 nM p/t and 100 nM of p66 Y232A /51 (Ⅺ) or p66 M230A /51 (f) or 300 nM of p66 W229A /51 (E). The reactions were stopped at a defined time point, and data were fitted to a hyperbola (see "Experimental Procedures"), which yielded K d values for dTTP as shown in Table IV for all enzymes. fails to yield heterodimeric RT (12). Crystallographic data from unliganded HIV-1 RT show that the side chains of Trp 229 , Met 230 , and Tyr 232 are oriented into the core region between the primer grip and the polymerase active site (22). We therefore assume that the mutations in Trp 229 , Gly 231 , or Leu 234 indirectly influence dimerization, for example by changing or preventing interactions with amino acids that contact the dimer interface. Fig. 7 shows possible interactions of the amino acids of the primer grip with surrounding residues as determined by the RT crystal structure of Rodgers et al. (22). A contact of Trp 229 with Pro 95 (distance ϳ3.79 Å) in the palm subdomain of p66 is possible by van der Waals interactions. Pro 95 is also involved in binding NNIs (19,20,23). Pro 95 in turn may interact with Asn 137 and Glu 138 in the finger subdomain of p51 (distance ϳ3.81 Å). By changing the bulky Trp 229 into Ala or by exchanging Leu 234 to Ala, a "deficiency of atoms" occurs in the pocket harboring these amino acids. This could lead to a conformational change of adjacent residues to fill the created space. As a consequence, the loop (residues 135-140) between ␤7 and ␤8 (shown in light purple in Fig. 7) of p51 containing Asn 137 and Glu 138 (highlighted in yellow in Fig. 7) and reaching into p66 is not able to contact the corresponding residues in p66 properly. Furthermore, this effect could lead to displacement of the ␤7-␤8 loop of p51 away from the pocket, thus destabilizing the dimer interface. At present we are investigating the role of this loop region of p51 in dimer formation. Interestingly, the only resistance mutation against NNIs found in p51 is located in this region (residue Glu 138 3 Lys), indicating a function of this loop in binding the NNI (24,25).
For residue Gly 231 , the situation might be somewhat different. It is located in the loop connecting ␤12 and ␤13 and therefore might have an important architectural role for the stability of the ␤12-␤13 hairpin. Exchanging this residue against the bulkier alanine could affect the stability of the hairpin, which in turn by a change in conformation of surrounding residues could have similar consequences for dimer stability as already described above.
Additionally, our results demonstrate that the conformational changes described influence the polymerization mechanism. Our experiments indicate a function for Trp 229 and Gly 231 in p/t binding and dNTP incorporation. Crystallographic data have implicated the primer grip in orienting the primer terminus for nucleophilic attack on an incoming dNTP (8). Our results with the mutants p66 W229A /51 and p66 G231A /51 imply that these enzymes are unable to align the primer properly, leading to a reduced affinity for p/t and dNTP and to a reduced incorporation rate for dNTP. However, the mutation in Met 230 clearly shows that the two effects, i.e. p/t binding and dNTP incorporation, can be separated. With mutant p66 M230A /51, dNTP binding and incorporation are impaired, but we do not find a significant reduction of the affinity for p/t DNA (Table  III). Modeling experiments that dock a dTTP into the polymerase active site of the HIV-1 RT/DNA/Fab cocrystal suggest that ␤-strand ␤6 (residues 105-118), the ␤9-␤10 hairpin (residues 178 -191), and the region ␤11a-␤11b (residues 214 -222) of p66 might be involved in dNTP binding (9,26). This allows positioning of the ␤and ␥-phosphate of the dTTP to the active site residues Asp 185 and Asp 186 . Direct interaction of the primer grip hairpin with a dNTP would be unfavorable, since the base of the dTTP in this case could not stack onto the terminal base of the primer. Therefore, it is reasonable to assume that the amino acids of the primer grip are not directly involved in dNTP binding. Instead, the changes in the orientation of the primer induced by the mutations probably lead to a less favorable positioning of the ␣-phosphate of the dNTP for attack by the 3Ј-OH of the primer terminus. However, minor changes of the primer orientation do not necessarily lead to a reduced affinity of the mutated enzyme for p/t but can have an affect on the stacking of the incoming nucleotide onto the terminal base of the primer. This is probably the case with mutant p66 M230A /51. This effect could also lead to a reduced fidelity of the mutant enzymes. In fact, preliminary results with mutant p66 W229A /51 indicate that the fidelity of this enzyme has also changed (data not shown).
Comparison of the results obtained for tRNA binding with those of p/t binding demonstrate that in the binary complex the primer grip region is not important for the affinity of RT for tRNA. We have shown previously by displacement titrations that a p/t DNA can be displaced by tRNA 3 Lys and various other tRNAs (14). This implies that tRNA and p/t bind to the same or at least partially overlapping sites. Together with the data presented here, these experiments suggest that the binding sites for tRNA and p/t are overlapping but not identical. A function of the primer grip in tRNA binding must be expected when the tRNA is hybridized to the primer binding site on the viral RNA, since here its 3Ј-OH end is used as a primer for the start of minus strand synthesis.
Previous results indicate that introduction of the mutation G231A into the virus leads to infectious particles in cell culture assays, whereas after introduction of W229A infectious virus is not formed (11,12). Our results show that in vitro both mutants are significantly impaired in polymerase function. The reason for this discrepancy is not clear, since most parameters we have measured for these two mutants (Tables I-IV) are similar. The only major difference (Table I) is in the steadystate activity assay, in which p66 W229A /51 is considerably worse than p66 G231A /51. This is initially surprising, since the single nucleotide incorporation kinetics of p66 W229A /51 are better than those of p66 G231A /51. We attribute this discrepancy to the poorer dimerization properties of p66 W229A /51, which probably lead to extensive dissociation under the low concentration conditions of the steady-state assay. In contrast, the quenched flow results were obtained at much higher protein concentration and are corrected for dimer content. We therefore conclude tentatively that impairment of heterodimer formation may be responsible for the lack of production of infectious virus for this mutant. Another possibility that has not been analyzed yet are compensatory mutations that could have occurred during the cell culture experiment to overcome the impairments of p66 G231A /51. In the absence of this, production of infectious virus after introduction of the G231A mutation remains a surprising result in view of the significantly impaired kinetic properties.
The observation that the mutation Y232A leads to a decrease in infectious particles in cell culture assays, although in in vitro assays the polymerization activity is not as drastically reduced as with mutant p66 G231A /51, is probably due to the impaired RNase H activity of p66 Y232A /51 demonstrated previously (12).
One general result of the work presented here is that single amino acid changes in HIV-1 RT can have far reaching and unexpected global effects on enzyme structure and function (e.g. dimerization), which can make interpretation of results difficult and misleading unless these factors are specifically taken into account. Such unexpected results can, however, be useful. As an example, the information obtained on the effects of mutations of the primer grip region on dimerization suggest that this region of the molecule might be included in the search for approaches to inhibition of dimerization.