Alanine-scanning Mutations in the ;Primer Grip; of p66 HIV-1 Reverse Transcriptase Result in Selective Loss of RNA Priming Activity
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.13262
ISSN1083-351X
AutoresMichael D. Powell, Madhumita Ghosh, Pamela S. Jacques, Kathryn J. Howard, Stuart F. J. Le Grice, JudithG. Levin,
Tópico(s)Hepatitis B Virus Studies
ResumoAlanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3′ end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3′ terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers. Alanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3′ end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3′ terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers. During the replicative cycle of human immunodeficiency virus (HIV) 1The abbreviations used are: HIV, human immunodeficiency virus; RT, reverse transcriptase; PBS, primer binding site; PPT, polypurine tract; WT, wild-type; nt, nucleotide(s). and other retroviruses, reverse transcriptase (RT) catalyzes the conversion of single-stranded genomic RNA into linear double-stranded DNA, which is ultimately integrated into the host chromosome (Ref. 1Gilboa E. Mitra S.W. Goff S. Baltimore D. Cell. 1979; 18: 93-100Abstract Full Text PDF PubMed Scopus (421) Google Scholar; for reviews, see Refs. 2Varmus H. Swanstrom R. Weiss R. Teich N. Varmus H. Coffin J. Molecular Biology of Tumor Viruses, RNA Tumor Viruses. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984: 369-512Google Scholar, 3Varmus H. Brown P. Berg D.E. Howe M.M. Mobile DNA. American Society for Microbiology, Washington, DC1989: 53-108Google Scholar, 4Arts E.J. Wainberg M.A. Adv. Virus Res. 1996; 46: 97-163Crossref PubMed Google Scholar). Reverse transcription begins with initiation of minus-strand DNA synthesis from a cellular tRNA primer (reviewed in Refs. 5Wilson S.H. Abbotts J. Hatfield D.L. Lee B.J. Pirtle R.M. Transfer RNA In Protein Synthesis. CRC Press, Inc., Boca Raton, FL1992: 1-21Google Scholar and 6Marquet R. Isel C. Ehresmann C. Ehresmann B. Biochimie. 1995; 77: 113-124Crossref PubMed Scopus (200) Google Scholar) bound to the primer binding site (PBS) at the 5′ end of the viral RNA (2Varmus H. Swanstrom R. Weiss R. Teich N. Varmus H. Coffin J. Molecular Biology of Tumor Viruses, RNA Tumor Viruses. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1984: 369-512Google Scholar). In this step, RT must recognize and extend an RNA primer annealed to an RNA template (7Leis J. Aiyar A. Cobrinik D. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 33-47Google Scholar,8Yusupova G. Lanchy J.-M. Yusupov M. Keith G. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. J. Mol. Biol. 1996; 261: 315-321Crossref PubMed Scopus (18) Google Scholar). Initiation of plus-strand DNA synthesis requires recognition of a purine-rich viral RNA sequence known as the polypurine tract (PPT). After specific RNase H cleavage at its 3′ end, the PPT serves as the primer for extension on a minus-strand DNA template (for review, see Ref. 9Champoux J.J. Skalka A.M. Goff S.P. Reverse Transcriptase. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 103-117Google Scholar). The specificity of this reaction is provided, in part, by the inability of RT to prime plus-strand synthesis with RNA fragments lacking the PPT sequence (10Huber H.E. Richardson C.C. J. Biol. Chem. 1990; 265: 10565-10573Abstract Full Text PDF PubMed Google Scholar, 11Randolph C.A. Champoux J.J. J. Biol. Chem. 1994; 269: 19207-19215Abstract Full Text PDF PubMed Google Scholar, 12Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Thus, to initiate synthesis of each DNA strand, RT specifically recognizes RNA primers, which have exacting sequence requirements and are extended on defined templates. In the case of HIV, formation of a specific (minus-strand) initiation complex between tRNALys3, genomic RNA, and RT is followed by transition to an elongation mode of synthesis (14Arts E.J. Li Z. Wainberg M.A. J. Biomed. Sci. 1995; 2: 314-321Crossref PubMed Scopus (12) Google Scholar, 15Isel C. Lanchy J.-M. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. EMBO J. 1996; 15: 917-924Crossref PubMed Scopus (178) Google Scholar, 16Lanchy J.-M. Ehresmann C. Le Grice S.F.J. Ehresmann B. Marquet R. EMBO J. 1996; 15: 7178-7187Crossref PubMed Scopus (109) Google Scholar). Elongation of minus-strand DNA involves extended interactions between the RNA template and tRNA primer (15Isel C. Lanchy J.-M. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. EMBO J. 1996; 15: 917-924Crossref PubMed Scopus (178) Google Scholar, 17Isel C. Ehresmann C. Keith G. Ehresmann B. Marquet R. J. Mol. Biol. 1995; 247: 236-250Crossref PubMed Scopus (229) Google Scholar) and also requires binding of RT to the 3′-OH of the growing DNA chain. As RT traverses the genomic RNA, it occasionally dissociates from the primer-template. To continue synthesis, it must therefore bind to different DNA primers on what is essentially a continuously-changing RNA template. Similarly, as elongation of plus-strand DNA proceeds, RT interacts with various DNA primers on a continuously changing DNA template. Thus, during elongation reactions, RT must be able to recognize DNA primers without regard to nucleotide sequence or configuration of the template. These considerations make it clear that at different steps in reverse transcription, RT is presented with primer-template combinations having different helical structure, geometry, and nucleic acid composition. In view of these variations, it is conceivable that RT may have evolved different mechanisms or structural features to selectively recognize RNA and DNA primers. Analysis of the x-ray crystal structure of HIV-1 RT complexed with a short duplex DNA primer-template indicates that the residues which interact with nucleotides at the 3′ end of the primer constitute the β12-β13 hairpin in the p66 palm known as the ;primer grip; (Fig. 1; Refs. 18Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1122) Google Scholar and 19Patel P.H. Jacobo-Molina A. Ding J. Tantillo C. Clark Jr., A.D. Raag R. Nanni R.G. Hughes S.H. Arnold E. Biochemistry. 1995; 34: 5351-5363Crossref PubMed Scopus (177) Google Scholar). Residues in the primer grip line one side of a hydrophobic pocket to which the non-nucleoside inhibitor nevirapine binds (Ref. 20Smerdon S.J. Jäger J. Wang J. Kohlstaedt L.A. Chirino A.J. Friedman J.M. Rice P.A. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3911-3915Crossref PubMed Scopus (357) Google Scholar; for review, see Ref. 21Tantillo C. Ding J. Jacobo-Molina A. Nanni R.G. Boyer P.L. Hughes S.H. Pauwels R. Andries K. Janssen P.A.J. Arnold E. J. Mol. Biol. 1994; 243: 369-387Crossref PubMed Scopus (495) Google Scholar). Interestingly, there are no nevirapine-resistant mutants with changes at residues Phe-227, Trp-229, and Leu-234. This suggests that these residues may be important for maintaining the structural integrity of the primer grip. Additionally, it has been shown that mutation of residues Trp-229, Met-230, Gly-231, and Tyr-232 results in alterations of both polymerase and RNase H activities (22Ghosh M. Jacques P.S. Rodgers D.W. Ottman M. Darlix J.-L. Le Grice S.F.J. Biochemistry. 1996; 35: 8553-8562Crossref PubMed Scopus (94) Google Scholar). In earlier work on the determinants of HIV-1 plus-strand priming, we showed that nucleotides at the 3′ end of the HIV-1 PPT primer are critical for initiation of plus-strand DNA synthesis (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Replacing the four G nucleotides at the 3′ end of the PPT with four C nucleotides, rendered the PPT completely inactive as a primer for plus-strand synthesis. In view of the proximity of the primer grip to the 3′ end of the primer (18Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1122) Google Scholar, 19Patel P.H. Jacobo-Molina A. Ding J. Tantillo C. Clark Jr., A.D. Raag R. Nanni R.G. Hughes S.H. Arnold E. Biochemistry. 1995; 34: 5351-5363Crossref PubMed Scopus (177) Google Scholar), these findings suggested that the primer grip may have a specific role in RT-catalyzed initiation of plus-strand DNA synthesis. The present study is focused on identification of residues that might be involved in unique interactions with the PPT primer. Our approach was to test the effect of introducing alanine substitutions within the primer grip on plus-strand priming. Our results show that mutations in this region can profoundly affect the ability of HIV-1 RT to extend an RNA PPT primer, while having little or no effect on priming with a DNA version of the PPT. Interestingly, most of these same mutations dramatically affect the ability of RT to initiate minus-strand DNA synthesis with an RNA oligonucleotide containing the 3′ 18 terminal nucleotides of the tRNALys3 primer (RNA PBS primer). With only one exception, these mutations do not have a significant effect on extension of a DNA PBS primer. Thus, it appears that residues in the primer grip region play a specific role in recognition and extension of RNA primers. RNA oligonucleotides were purchased from Oligos Etc., Inc. (Wilsonville, OR). Other materials were used as described previously (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar, 22Ghosh M. Jacques P.S. Rodgers D.W. Ottman M. Darlix J.-L. Le Grice S.F.J. Biochemistry. 1996; 35: 8553-8562Crossref PubMed Scopus (94) Google Scholar). Alanine substitutions in residues Glu-224 to His-235 in HIV-1 RT were constructed using BcgI cassette mutagenesis as described previously (23Jacques P.S. Wöhrl B.M. Ottmann M. Darlix J.-L. Le Grice S.F.J. J. Biol. Chem. 1994; 269: 26472-26478Abstract Full Text PDF PubMed Google Scholar). The p66 subunits of mutant RTs were expressed separately and reconstituted with wild-type (WT) p51 to form p66/p51 heterodimers; thus, the mutation in each RT was present solely in the p66 subunit (24Le Grice S.F.J. Grüninger-Leitch F. Eur. J. Biochem. 1990; 187: 307-314Crossref PubMed Scopus (300) Google Scholar, 25Le Grice S.F.J. Naas T. Wohlgensinger B. Schatz O. EMBO J. 1991; 10: 3905-3911Crossref PubMed Scopus (190) Google Scholar). Reconstituted heterodimers were purified by metal chelate (Ni2+-nitrilotriacetic acid-Sepharose) chromatography followed by ion exchange over S-Sepharose (26Le Grice S.F.J. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-147Crossref PubMed Scopus (121) Google Scholar). The L234A mutant RT failed to reconstitute into a heterodimer (22Ghosh M. Jacques P.S. Rodgers D.W. Ottman M. Darlix J.-L. Le Grice S.F.J. Biochemistry. 1996; 35: 8553-8562Crossref PubMed Scopus (94) Google Scholar) and was not studied further. The final RT preparations were stored in a 50; glycerol-containing buffer at −20 °C (26Le Grice S.F.J. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-147Crossref PubMed Scopus (121) Google Scholar). The ability of each RT to initiate plus-strand DNA synthesis was tested in an assay using synthetic RNA and DNA oligonucleotides, as described previously (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Four primer-template combinations (Table I) were used (see schematic representation at the bottom of Fig. 2). The templates were all 35-nt DNA oligonucleotides. The primers were as follows. (i) A 15-nt RNA PPT already containing the 3′ end normally generated after specific cleavage by the RNase H activity of WT RT was used to test the ability of RT to extend the PPT without requiring prior cleavage. (ii) A 20-nt RNA PPT containing the PPT sequence and the five bases immediately downstream of the PPT was used to test the ability of RT to specifically cleave the PPT prior to extension. (iii) Downstream RNA, a non-priming sequence was used. This RNA oligonucleotide contains the 15-nt sequence immediately downstream of the PPT. It does not function as a primer with WT HIV-1 RT (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar) and serves as a negative control. (iv) A 15-nt DNA version of the PPT was used to test for primer extension with a DNA primer. Each reaction contained 1 pmol of primer-template and 10 pmol of WT or mutant HIV-1 RT. The products were internally labeled by addition of [α-32P]dATP during synthesis. The reactions (total volume, 15 μl) were carried out at 37 °C for 15 min and were terminated by addition of formamide STOP solution. Reaction products were then heated to 95 °C for 5 min prior to loading on an 8; sequencing gel and visualization by autoradiography.Table IOligonucleotides used in this studyDesignationSequenceType15-nt PPT5′-AAAAGAAAAGGGGGGRNA20-nt PPT5′-AAAAGAAAAGGGGGGACUGGRNADNA PPT5′-AAAAGAAAAGGGGGGDNAPPT template3′-TTTTCTTTTCCCCCCTGACCTTCCCGATTAAGTGADNABand shift template3′-TTTTCTTTTCCCCCCTGADNADownstream RNA5′-ACUGGAAGGGCUAAURNADownstream template3′-TGACCTTCCCGATTAAGTGAGGGTTGCTTCTGTTCDNARNA PBS primer5′-GUCCCUGUUCGGGCGCCARNADNA PBS primer5′-GTCCCTGTTCGGGCGCCADNA Open table in a new tab Catalytic rate constants for primer extension with a 15-nt RNA or DNA PPT primer were determined for WT RT and mutants E224A, P225A, and L228A. The time course for RNA PPT primer extension was performed essentially as described under ;Oligonucleotide Assays for Initiation of Plus-strand DNA Synthesis,; except that the reaction volume was increased 3-fold to 45 μl and 5-μl aliquots were removed at 1, 2, 3, 4, 5, 6, 10, and 15 min. Each 5-μl aliquot was added to 2 μl of formamide STOP solution. Since primer extension with a DNA PPT was very rapid (<1 min to reach completion) under the conditions of enzyme excess used in our standard assay, it was necessary to dilute each enzyme by 1:50 (0.2 pmol); this results in a molar ratio of enzyme to primer-template of 1:5. Dilutions of RT greater than 1:50 were not used since low enzyme concentrations are associated with an increase in pausing (data not shown). Reactions were carried out as described above with the RNA PPT primer. However, in reactions with WT RT and the DNA PPT primer, time points were also taken at 15, 30, and 45 s to provide a more accurate curve-fit. Samples were analyzed on an 8; sequencing gel, as described under ;Oligonucleotide Assays for Initiation of Plus-strand DNA Synthesis.; The amount of radioactivity incorporated into the 20-nt DNA product for the RNA PPT or the 35-nt product for the DNA PPT was determined by phosphorimaging, using the Molecular Dynamics STORM system. Values were converted to picomoles incorporated by comparison of the counts at each time point to counts from a control reaction containing WT RT incubated with the DNA PPT primer for 15 min (under standard assay conditions). Under these conditions, maximal incorporation is achieved in <1 min. Since 1 pmol of template is used in each reaction and each template is extended by 20 nt, the total incorporation is 20 pmol of nucleotides. The values from three independent experiments were averaged and then plotted as picomoles of nucleotides incorporated versus time in min. The resulting data were then fit to a single exponential equation, using the general curve-fitting routines found in the Macintosh program KaleidaGraph. Rate constants were determined as described by Beard and Wilson (27Beard W.A. Wilson S.H. Biochemistry. 1993; 32: 9745-9753Crossref PubMed Scopus (49) Google Scholar), based on a model for DNA polymerase I developed by Bryant et al. (28Bryant F.R. Johnson K.A. Benkovic S.J. Biochemistry. 1983; 22: 3537-3546Crossref PubMed Scopus (150) Google Scholar). Since the rate constants for the RNA PPT primer extension were determined under conditions of 10-fold enzyme excess and represent the relatively slow rate of initiation, the values are independent of enzyme concentration. In this case, the rates are expressed only as the derived rate constants from the exponential fit. However, initiation was not as slow for DNA PPT-primed extension and the reaction conditions had to be altered (see above). In this case, primer-template is in excess and the rate constants represent the apparent turnover rates (k =v i/[RT]), where v i is the initial rate determined from the curve-fit and [RT] is enzyme concentration in picomoles. Binding to primer-template duplexes was investigated with two types of bandshift assays, essentially as described by Guo et al. (29Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar). (i) The first type was binding only. One pmol (∼2 × 104 total counts per min) of 5′ end labeled ;band-shift DNA template; (Table I) was annealed to 10 pmol of the 15-nt RNA or DNA PPT primers. Binding conditions for the band-shift assay were the same as those used in the oligonucleotide assay, except that dNTPs and [α-32P]dATP were omitted. After incubation at 37 °C for 15 min, glycerol was added to a final concentration of 20; (v/v) and 10-μl portions of each reaction were loaded onto a 6; native polyacrylamide gel. Electrophoresis was carried out at room temperature for 60 min at a constant voltage of 200 V in a buffer containing 25 mm Tris-HCl and 162 mm glycine, pH 8.0. (ii) The second type was binding plus extension. Binding and extension were tested in the same band-shift assay by incubating an unlabeled DNA PPT primer-template (Table I) under the conditions used in the oligonucleotide assay, except that [α-32P]dATP was the only dNTP added. The first base downstream from the PPT is a thymidine (see Table I). Thus, in this assay only primer-template that has been extended by one base will be labeled (29Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar). Since most of the mutant RTs were unable to extend the RNA PPT primer, an assay was developed to test for the ability to catalyze specific cleavage at the 3′ terminus of the PPT intrans. The 15-nt PPT primer was annealed to the PPT template (Table I) and extended using T4 DNA polymerase and internal labeling with [α-32P]dATP, as described previously (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Where specified, 10 pmol of WT or mutant RT was then added intrans and incubated with 1 pmol of substrate in a final volume of 15 μl for 15 min at 37 °C. The products were analyzed by polyacrylamide gel electrophoresis, as described above under ;Oligonucleotide Assays for Initiation of Plus-strand DNA Synthesis.; The ability of each mutant to initiate minus-strand strong-stop DNA synthesis was tested using a PBS-containing RNA template (30Arts E.J. Ghosh M. Jacques P.S. Ehresmann B. Le Grice S.F.J. J. Biol. Chem. 1996; 271: 9054-9061Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) annealed to either an RNA or DNA primer which is complementary to the PBS. The conditions used were the same as those previously described (22Ghosh M. Jacques P.S. Rodgers D.W. Ottman M. Darlix J.-L. Le Grice S.F.J. Biochemistry. 1996; 35: 8553-8562Crossref PubMed Scopus (94) Google Scholar), except that the enzyme concentration was increased to achieve a 10:1 molar ratio of RT to primer-template. Synthesis was allowed to proceed for 60 min at 37 °C. Production of full-length strong-stop DNA on this RNA template results in a 192-nt DNA product (see Fig. 7). In an earlier study, we developed a simple oligonucleotide assay to test for the ability of HIV-1 RT to initiate plus-strand DNA synthesis from the 3′ PPT (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). We used this assay to screen alanine-scanning mutants of the primer grip (residues 224–235; Fig. 1; Ref. 18Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1122) Google Scholar) with four different primer-template combinations (see Table I and the bottom of Fig. 2): (i) a 15-nt RNA PPT primer, to measure specific extension; (ii) a 20-nt RNA PPT primer, to measure specific cleavage and subsequent extension; (iii) a non-PPT containing RNA oligonucleotide, which serves as a negative control; and (iv) a 15-nt DNA version of the PPT. The results are shown in Fig. 2 as lanes 1–4, respectively, for each enzyme. WT HIV-1 RT readily extended both the 15- and 20-nt RNA PPT oligonucleotides to produce a specific 20-nt plus-strand DNA product (Fig. 2, WT, lanes 1 and 2 and the schematic shown below). In the case of the 20-nt primer, cleavage of the five additional downstream bases occurs prior to extension (13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). As previously observed (10Huber H.E. Richardson C.C. J. Biol. Chem. 1990; 265: 10565-10573Abstract Full Text PDF PubMed Google Scholar, 11Randolph C.A. Champoux J.J. J. Biol. Chem. 1994; 269: 19207-19215Abstract Full Text PDF PubMed Google Scholar, 12Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar), HIV-1 RT was unable to extend an RNA primer consisting of a non-PPT containing downstream sequence (Fig. 2,WT, lane 3). A DNA version of the PPT was efficiently extended, forming a 35-nt product consisting solely of DNA (Fig. 2, WT,lane 4). When we tested the primer grip mutants using these same assays, we found that 8 of the 11 mutants (i.e. P226A, F227A, W229A, M230A, G231A, Y232A, E233A, and H235A) lost the ability to extend an RNA PPT primer (Fig. 2, lanes 1 and 2 for each enzyme). In the case of P226A, a very small amount (<5; of WT) of plus-strand DNA product was detectable. All mutants, with the exception of W229A, extended a DNA version of the PPT primer as efficiently as WT under standard assay conditions (Fig. 2, lane 4 for each enzyme). The W229A mutant was previously reported to have defective polymerase activity with a primer-template containing non-viral sequences (23Jacques P.S. Wöhrl B.M. Ottmann M. Darlix J.-L. Le Grice S.F.J. J. Biol. Chem. 1994; 269: 26472-26478Abstract Full Text PDF PubMed Google Scholar). Thus, it appears that mutations at positions 226, 227, 229, 230–233, and 235 have a specific effect on recognition of the RNA PPT primer. As is the case for the WT enzyme, none of the mutants was able to extend the non-PPT containing RNA oligonucleotide (lane 3 in each set; Ref. 13Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar). Changes in reaction conditions such as an increase in incubation time (up to 60 min) or concentration of the enzymes (up to a 60:1 ratio of RT to primer-template) had no effect on the amount of plus-strand product made by mutants that were inactive in the standard assay (data not shown). This suggests that the specific defect in ability of these RTs to recognize the RNA PPT is not kinetic in nature, but instead represents a loss in ability to add a base to the primer terminus. Mutants E224A, P225A, and L228A behaved essentially like WT HIV-1 RT in our standard end point assay (Fig. 2, lanes 1–4 ofE224, P225, and L228). However, kinetic analysis of priming activities with the 15-nt RNA PPT primer revealed that the catalytic rate constants determined for mutants P225A and L228A were approximately 4- and 6.5-fold lower, respectively, than the value for WT RT, k = 0.52 min−1; the rate constant for mutant E224A was very similar to that for the WT (Fig. 3 A). Results from this analysis also revealed that, although the overall time for completion of the reaction is relatively long (on the order of min), no intermediate-size products were observed (data not shown). This suggests that once the RNA primer is extended, subsequent elongation and cleavage must occur very rapidly. Similar analysis of the time course of primer extension with the 15-nt DNA PPT primer indicated that under our standard conditions where RT is in excess, reactions with mutants E224A, P225A, L228A, and WT RT proceeded too rapidly for determination of rate constants (data not shown). Thus, for these determinations, we modified the assay conditions and reduced the amount of each enzyme by 50-fold; this results in a 1:5 ratio of enzyme to primer-template. The catalytic rates for each of the mutant enzymes with the DNA PPT primer were similar (k ≅ 1.30 min−1) and were approximately 4-fold lower than that determined for WT RT (Fig.3 B). To test the possibility that the primer grip mutants which could not prime plus-strand DNA synthesis with the RNA PPT are unable to bind to unextended primer-template, we performed band-shift assays (29Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar) using the RNA and DNA PPTs as primers (Fig.4). We tested each mutant for the ability to form a stable complex under the same conditions used in the standard assay for plus-strand DNA synthesis (i.e. at a 10:1 ratio of RT to primer-template). All of the primer grip mutants were able to form at least small amounts of a stable complex with the RNA PPT primer-template, although the fraction of shifted complex obtained with the mutant RTs was not as great as that seen with WT RT (Fig. 4 A). In the case of mutant Y232A, the complex was barely detectable (Fig.4 A). Interestingly, variation in binding of the different mutant RTs to the RNA PPT primer-template did not strictly correlate with their ability to extend this primer. For example, mutants E224A and P225A could extend the RNA PPT (Fig. 2), but the extent of binding seen with these RTs was similar to that of F227A (Fig. 4 A), which could not extend the RNA PPT (Fig. 2). The reduction in binding efficiency of the mutants to the DNA PPT primer-template was similar to that found with the hybrid containing the RNA PPT (Fig. 4 B). In addition, when the ratios of enzyme to primer-template were increased above 10:1 for these mutants, most of the RNA primer-template could ultimately be shifted to a stable complex (data not shown). Thus, it appears that the defect in the ability to extend the RNA PPT is not simply a loss of ability to bind to the primer-template, but a loss in the ability to extend the bound primer. Some mutants (i.e. P226A, M230A, G231A, Y232A, and H235A; Fig. 4 B) showed relatively poor binding to the DNA PPT primer-template but were able to efficiently extend the DNA PPT primer (see Fig. 2). To investigate a possible correlation between the amount of stable complex formed in the band-shift assay with the ability to prime from the DNA PPT, we performed the band-shift assay under conditions in which binding and extension were tested in the same experiment (Fig. 5). The experimental conditions were the same as those used in the DNA PPT band-shift experiment (Fig.4 B), except that the primer-template was unlabeled and [α-32P]dATP was included to allow labeling by incorporation of dA at the +1 position. In this case, the primer-template is labeled only if the enzyme is able to bind to an extent necessary for incorporation of a single, labeled base (29Guo J. Wu W. Yuan Z.Y. Post K. Crouch R.J. Levin J.G. Biochemistry. 1995; 34: 5018-5029Crossref PubMed Scopus (41) Google Scholar). More pronounced differences between the primer grip mutants were observed with this assay. While all of the label incorporated by WT RT was present in bound primer-
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