Mutational Analysis of Tyr-501 of HIV-1 Reverse Transcriptase

N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg2+ or Mn2+) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or π-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication “fitness” of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and inN-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development. N-(4-tert-Butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH) is a potent inhibitor of the ribonuclease H (RNase H) activity of human immunodeficiency virus (HIV)-1 reverse transcriptase (RT). Molecular modeling predicted that BBNH binds to the HIV-1 RT RNase H active site via two major interactions, coordination to the metal ion cofactor (Mg2+ or Mn2+) in the enzyme active site and aromatic ring-stacking interaction between the naphthyl ring of BBNH and amino acid Tyr-501. The latter residue equivalent is conserved in virtually all RNases H, suggesting the need for an aromatic or π-stacking interaction in this region. To assess the importance of Tyr-501 in the binding of BBNH for the inhibition of RT RNase H activity, we used site-specific mutagenesis to generate RT with a variety of substitutions at this position. Most substitutions resulted virtually in a complete loss of RNase H activity. However, three mutants, Y501F, Y501W, and Y501R, possessed RNase H activities comparable with wild-type enzyme. Whereas BBNH inhibited Y501F RT RNase H activity with potency equivalent to wild-type RT, the Y501W mutant showed a 6-fold resistance to inhibition by BBNH, and the Y501R mutant was completely resistant to inhibition by BBNH. The replication “fitness” of HIV molecular clones with the Y501W or Y510R mutation was significantly compromised compared with wild-type virus. Importantly, BBNH was an effective inhibitor of the DNA polymerase activity of all Y501X mutants tested. Our results highlight the importance of Tyr-501 in RT RNase H activity and inN-acylhydrazone inhibitor binding and suggest that drugs that target critical residues in HIV-1 proteins may be a useful approach in new antiviral development. reverse transcriptase ribonuclease H N-(4-tert-butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone N-(4-tert-butylbenzoyl)-2-hydroxy-1-salicylylhydrazone human immunodeficiency virus type 1 non-nucleoside inhibitor binding pocket wild type The conversion of retroviral genomic RNA into the double-stranded viral DNA intermediate is catalyzed entirely by the viral enzyme, reverse transcriptase (RT).1To carry out this complex conversion, RT must be multifunctional, possessing RNA-dependent DNA polymerase activity to synthesize DNA complementary to the viral genomic RNA, ribonuclease H (RNase H), activity in order to degrade the RNA component of the resultant RNA-DNA duplex, thereby releasing the newly formed DNA as a template to allow the DNA-dependent DNA polymerase activity to complete the synthesis of double-stranded viral DNA. RT RNase H is also crucial for the strand transfer reactions that are essential for the synthesis of viral DNA. Because of the essential role of HIV-1 RT in virus replication, numerous anti-chemotherapeutic agents have been developed against this enzyme (for recent reviews, see Refs. 1Parniak M.A. Sluis-Cremer N. Adv. Pharmacol. 2000; 49: 67-109Google Scholar and 2Sluis-Cremer N. Arion D. Parniak M.A. Cell. Mol. Life Sci. 2000; 57: 1408-1422Google Scholar). Virtually all of these inhibitors are directed against RT DNA polymerase activity with very few inhibitors of RT RNase H so far identified. The few inhibitors of HIV RT RNase H include illimaquinone (3Loya S. Tal R. Kashman Y. Hizi A. Antimicrob. Agents Chemother. 1990; 34: 2009-2012Google Scholar), azidothymidylate (4Tan C.-K. Civil R. Mian A.M. So A.G. Downey K.M. Biochemistry. 1991; 30: 4831-4835Google Scholar, 5Zhan X. Tan C.-K. Scott W.A. Mian A.M. Downey K.M. So A.G. Biochemistry. 1994; 33: 1366-1372Google Scholar), certain naphthalenesulfonic acid derivatives (6Mohan P. Loya S. Avidan O. Verma S. Dhindsa G.S. Wong M.F. Huang P.P. Yashiro M. Baba M. Hizi A. J. Med. Chem. 1994; 37: 2513-2519Google Scholar), N-ethylmaleimide (7Loya S. Gao H.-Q. Avidan O. Boyer P.L. Hughes S.H. Hizi A. J. Virol. 1997; 71: 5668-5672Google Scholar), poly(1-methyl-6-thioinosinic acid) (8Buckheit Jr., R.W. Lackman-Smith C. Snow M.J. White E.L. Ross L.J. Agrawal V.K. Broom A.D. Antiviral Chem. Chemother. 1999; 1: 23-32Google Scholar), and certain plant or stem-bark extracts (9Hnatyszyn O. Broussalis A. Herrera G. Muschietti L. Coussio J. Martino V. Ferraro G. Font M. Monge A. Martinez-Irujo J.J. Sanroman M. Cuevas M.T. Santiago E. Lasarte J.J. Phytother. Res. 1999; 13: 206-209Google Scholar, 10Min B.S. Nakamura N. Miyashiro H. Kim Y.H. Hattori M. Chem. Pharm. Bull. (Tokyo). 2000; 48: 194-200Google Scholar). None of these is especially potent inhibitors. To date, the only compounds shown to inhibit HIV-1 RT RNase H with reasonable potency areN-acylhydrazones (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar) and phenylhydrazones such as PD029687 (12Gabbara S. Davis W.R. Hupe L. Hupe D. Peliska J.A. Biochemistry. 1999; 38: 13070-13076Google Scholar). We were the first group to describe a reasonably potent inhibitor of HIV-1 RT RNase H activity (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). This inhibitor,N-(4-tert-butylbenzoyl)-2-hydroxynaphthaldehyde hydrazone (BBNH, Fig. 1), is one of a family of metal-binding hydrazones, which were developed primarily as antitumor agents and antioxidants (13Baker E. Richardson D. Gross S. Ponka P. Hepatology. 1992; 15: 492-501Google Scholar). BBNH inhibits both the HIV-1 RT DNA polymerase and RNase H domains (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). BBNH also inhibits the RNase H activity of HIV-2 RT, Moloney murine leukemia virus RT, andEscherichia coli RNase HI (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). We have suggested that this multifunctional inhibition is the result of the binding of two molecules of BBNH/molecule of HIV-1 RT with one molecule interacting close to the non-nucleoside RT inhibitor-binding pocket (NNIBP) in the polymerase domain of the enzyme and the second molecule binding in the spatially distinct RNase H domain of the enzyme (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). Molecular modeling suggested that BBNH binds to the RT RNase H domain close to the active site, and that residue Tyr-501 is integral in the binding interaction. We therefore altered Tyr-501 by site-directed mutagenesis to better define this potential interaction. Our results indicate that this residue appears to be essential for RT RNase H activity, because most substitutions abrogate enzyme activity. The Y501F, Y501W, and Y501R mutants retained appreciable RNase H activity, and only the Y501F mutant remained sensitive to BBNH inhibition, an observation consistent with the molecular model for BBNH binding to the RNase H domain. Importantly, the introduction of the Y501W and Y501R mutations in molecular clones of HIV-1 resulted in virus that was significantly attenuated in replication fitness. BBNH andN-(4-tert-butylbenzoyl)-2-hydroxy-1-salicylylhydrazone (BBSH) were synthesized by standard methods (14Edward J.T. Gauthier M. Chubb F.L. Ponka P. J. Chem. Eng. Data. 1988; 33: 538-540Google Scholar). 1H NMR and elemental analyses of these compounds were entirely consistent with the expected chemical structures. [3H]TTP, [γ-32P]ATP, and the homopolymeric template/primer poly(rA)-oligo(dT)12–18 were purchased from Amersham Biosciences, Inc. All other reagents were of the highest quality available and were used without further purification. A model for the interaction of BBNH with HIV-1 RT was constructed using the x-ray crystallographic coordinates for HIV-1 RT complexed with nevirapine (15Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Google Scholar) (Protein Data Bank accession number 3HVT). This structure was chosen because of the presence of magnesium atoms bound in the RNase H active site. The initial conformation of BBNH was derived from its x-ray crystal structure (16Lanthier C.M. Parniak M.A. Dmitrienko G.I. Bioorg. Med. Chem. Lett. 1997; 7: 1557-1562Google Scholar). Docking and energy minimization experiments were carried out with the anneal function of Sybyl 6.5 (Tripos Inc., St Louis, MO). Charges were calculated by the Gasteiger-Huckel method, and iterative minimization was carried out with the Tripos force field until the energy difference between iterations was <0.01 kcal/mol/Å. The p66- and p51-kDa subunits of HIV-1 RT were cloned into a pBAD/HisB prokaryotic expression vector (Invitrogen) between the XhoI and HindIII restriction endonuclease cleavage sites. Mutations at position 501 were introduced into the gene encoding for the p66-kDa subunit of HIV-1 RT by site-directed mutagenesis using either the SculptorTMin vitro mutagenesis (Amersham Biosciences, Inc.) or QuikChangeTM site-directed mutagenesis (Stratagene) systems. All clones were sequenced for the desired mutation using a T7-sequencing kit (Amersham Biosciences, Inc.). WT and mutant RT p66/p51 heterodimers were purified as described previously (17Fletcher R.S. Holleshak G. Nagy E. Arion D. Borkow G. Gu Z. Wainberg M.A. Parniak M.A. Protein Expression Purif. 1996; 7: 27-32Google Scholar). The specific activities of WT and mutant RT were determined by measuring the RNA-dependent DNA polymerase activity of each enzyme using 0.2 units/ml of poly(rA)-oligo(dT)12–18, 20 μm[3H]dTTP, and 8.5 nm purified RT p66/p51 heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mmKCl, 10 mm MgCl2. Samples were incubated for 10 min at 37 °C and then quenched by the addition of ice-cold 10% trichloroacetic acid containing 20 mm sodium pyrophosphate. After a 20-min incubation on ice, samples were filtered using a 1.2-μm glass fiber type C filter multi-well plates (Millipore) and washed sequentially with 10% trichloroacetic acid and ethanol. The extent of radionucleotide incorporation was then determined by liquid scintillation spectrometry. Assays for the inhibition of RT DNA polymerase activity by BBNH were carried out as described previously (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). More detailed inhibition kinetic analysis of RT RNA-dependent DNA polymerase activity employed a heteropolymeric template/primer prepared as previously described (18Arts E.J. Li X. Gu Z. Kleiman L. Parniak M.A. Wainberg M.A. J. Biol. Chem. 1994; 269: 14672-14680Google Scholar) using the T7 polymerase RNA transcript fromAccI-linearized plasmid pHIV-PBS as template and a synthetic 18-nucleotide deoxyoligonucleotide as DNA synthesis initiation primer. Reaction assays comprised 0.5 units/ml of heteropolymeric template/primer, variable concentrations of a mixture of equimolar [3H]dATP, [3H]dCTP, [3H]dGTP, [3H]TTP (ranging from 1–20 μm total dNTP concentration), and 10 nmpurified RT p66/p51 heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 10 mm MgCl2. Samples were incubated and processed as described above. Inhibition data were analyzed by Dixon plot analysis. RNase H activity was assayed using an 18-nucleotide 5′-32P-heteropolymeric RNA template (5′-GAUCUGAGCCUGGGAGCU-3′) annealed to a complementary 18-nucleotide DNA oligomer (5′-AGCTCCCAGGCTCAGATC-3′). 14 nm 5′-[32P]RNA-DNA duplex was incubated with 5 nm RT heterodimer in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 2.5 mm MgCl2 in 10 μl of final volume. Samples were incubated for 10 min at 37 °C and then quenched with an equal volume of gel loading dye (98% deionized formamide, 10 mm EDTA, 1 mg/ml bromphenol blue, and 1 mg/ml xylene cyanol). RNase H-generated degradation products were resolved by electrophoresis using 14% acrylamide/7 m urea sequencing gels visualized by autoradiography and quantified by densitometry. For assay of BBNH or BBSH inhibition of RT RNase H activity, WT or mutant RT (30 nm) was preincubated with BBNH or BBSH (dissolved in Me2SO) in 50 mm Tris-HCl, pH 7.9, 60 mm KCl, 2.5 mm MgCl2, 0.2 mg/ml of ovalbumin in a final volume of 50 μl for 10 min. The final concentration of Me2SO never exceeded 2%. 10-μl aliquots of the preincubation mixture was then added to 40 μl of an assay mixture containing 14 nm RNA-DNA duplex and assayed as described above. Inhibition data were calculated from Dixon and Lineweaver-Burk plots. Plasmid SVC21-BH10 encodes an infectious molecular clone of the IIIB strain of HIV-1 and carries an SV40 origin of replication for expression in COS cells (19Fisher A.G. Collati E. Ratner L. Gallo R.C. Wong-Staal F. 1991; Scholar). Mutations in the to residue 501 of the RT gene were introduced using the mutagenesis kit was prepared by of cells using a J. Molecular A Scholar). were and the of virus in these was quantified by the of HIV-1 of these containing equal of viral were then added to of were for the of aliquots of were for the of viral The model for the binding of BBNH to the RNase H domain of HIV-1 RT is shown in Fig. analysis BBNH to be a molecule (16Lanthier C.M. Parniak M.A. Dmitrienko G.I. Bioorg. Med. Chem. Lett. 1997; 7: 1557-1562Google Scholar). Our indicate a between the inhibitor and a close to the RNase H active site that is and to binding of the bound inhibitor include and Tyr-501 major binding were identified. The first the coordination of one of the atoms and the hydrazone to the RNase H active site This interaction is consistent with that BBNH inhibition of RT RNase H activity is a metal coordination is and that BBNH is a inhibitor of RT RNase H activity (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). The second major interaction is an interaction between the ring of Tyr-501 and the second naphthyl ring of BBNH To this we the potency of a BBNH 1), in which a aromatic ring the naphthyl ring of BBNH. The ring is to in aromatic with and in with model was an inhibitor of RT RNase H activity BBSH retained potency against RT DNA polymerase of the DNA polymerase and RNase H activities of HIV-1 RT by BBNH and are the from three each carried out in For of RT RNA-dependent DNA polymerase activity. were carried out using as template/primer, and were using a heteropolymeric template/primer as described Inhibition for RNase H activity used the heteropolymeric duplex as described in a new are the from three each carried out in For of RT RNA-dependent DNA polymerase activity. were carried out using as template/primer, and were using a heteropolymeric template/primer as described Inhibition for RNase H activity used the heteropolymeric duplex as described To the interaction between Tyr-501 and BBNH, we a number of mutant with substitutions at position Y501F, Y501W, Y501R, and Most of these mutant significantly RNase H activity and also showed attenuated RNA-dependent DNA polymerase activity compared with the WT enzyme. the Y501F, Y501W, and Y501R mutants retained appreciable RNase H RNA-dependent DNA polymerase and RNase H activities of Y501X mutant HIV-1 specific wild-type specific activity of wild-type p66/p51 RT was determined with as template/primer as described were determined by of the in the of 18-nucleotide to complementary DNA as described The specific activity of wild-type p66/p51 RT was determined with as template/primer as described were determined by of the in the of 18-nucleotide to complementary DNA as described in a new BBNH inhibited the RNase H activity of Y501F RT as as WT RT However, the Y501W mutant showed a 6-fold to inhibition of its RNase H activity by BBNH, and the RNase H activity of the Y501R mutant enzyme was completely resistant to BBNH the DNA polymerase activity of all remained sensitive to inhibition by BBNH inhibition of the and RNase H activities of Y501X mutants of HIV-1 are the from three each carried out in were carried out using as template/primer, and were using a heteropolymeric template/primer as described are the from three each carried out in because these mutants RNase H activity in the of are the from three each carried out in were carried out using as template/primer, and were using a heteropolymeric template/primer as described are the from three each carried out in in a new because these mutants RNase H activity in the of Molecular clones of HIV-1 with substitutions at RT residue 501 were constructed and for to and in the Y501F and Y501W mutant were to as the Y501R However, the mutants showed replication to WT virus of HIV-1 with mutations at residue 501 of are from virus was are from in a new virus was We previously suggested that the of BBNH to inhibit RT activities be because of the binding of the inhibitor to two on the enzyme on and kinetic data (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). Our was that binding to a site at or the in the polymerase domain of RT results in the inhibition of the DNA polymerase activity of the enzyme, binding to the second site in the RNase H domain inhibition of the RNase H activity of the enzyme. However, was that the inhibition of both activities from site binding of the certain non-nucleoside reverse transcriptase inhibitors have been shown to RT RNase H activity binding only to the V. S. J. Biol. Chem. 1994; 269: Scholar). Molecular modeling showed that BBNH be into the RT RNase H domain in a consistent with previously data For a major interaction the coordination of the of BBNH with the active site metal in the RNase H domain. This is consistent with the metal for BBNH inhibition of RT RNase H activity as as the of this inhibition (11Borkow G. Fletcher R.S. Barnard J. Arion D. Motakis D. Dmitrienko G.I. Parniak M.A. Biochemistry. 1997; 36: 3179-3185Google Scholar). very few potent molecule inhibitors of HIV-1 RT RNase H have been identified. This may be to the that the RNase H active site a structure be expected for an active site that must the RNA-DNA duplex and appears that the potential to allow for reasonable binding with However, BBNH is a molecule with an structure (16Lanthier C.M. Parniak M.A. Dmitrienko G.I. Bioorg. Med. Chem. Lett. 1997; 7: 1557-1562Google Scholar) and metal-binding that allow interaction with the active site the only other potent inhibitor of RT RNase H, the was shown to also be of metal metal binding may be an for the of RNase H inhibitors. The molecular model also that aromatic ring-stacking between the ring of Tyr-501 and the second ring of the naphthyl of BBNH is for the binding of the inhibitor with the RT RNase H active site. The observation that a BBNH with a ring is to inhibit RT RNase H is also consistent with this BBSH a potent inhibitor of RT DNA polymerase activity, that this to the RT polymerase domain at or the This observation further that the inhibition of RT activities by BBNH from the binding of the inhibitor to on analyses of Tyr-501 also the that this residue is crucial for the binding of BBNH. the of bound acid duplex Tyr-501 is completely to and the ring is to its This the of Tyr-501 to an to with the naphthyl ring of BBNH, thereby the crucial interaction. The Y501F the the RNase H activity of Y501F RT is sensitive to BBNH The of the Y501W mutant the the This the from the to BBNH consistent with the of the Y501W mutant to BBNH Whereas is of interactions, the and of the Y501R mutation is such that with BBNH be the RNase H activity of the Y501R mutant RT is completely to BBNH Importantly, the DNA polymerase activity of the Y501F, Y501W, and Y501R mutants sensitive to inhibition by BBNH, that the inhibition of RT activities of WT enzyme from site binding of the Our analyses of Tyr-501 also the observation that most substitutions at this position resulted in of RNase H activity, suggesting an essential role for Tyr-501 in RT RNase H activity. this is to that virtually all RNases H for which is available have a residue in the position to that of Tyr-501 in the HIV-1 RT RNase H domain C.M. Protein Sci. 7: Scholar, J.J. H. Scholar). The role of Tyr-501 is at is to that each of the amino acid substitutions at which of enzyme activity, and are of in π-stacking is that Tyr-501 in with of the RNA-DNA acid duplex thereby in the in the RNase H active site. crystal of the and of HIV-1 RT bound with double-stranded DNA suggest that Tyr-501 the bound acid duplex J. Y. A.D. A. C. Hughes S.H. E. J. Mol. Biol. Scholar, H. R. Science. Scholar). However, further is to the role of Tyr-501 in RT RNase H and the equivalent residue in other RNases H. the Y501F, Y501W, and Y501R mutant RT retained WT of DNA polymerase and RNase H activity in in vitro molecular clones of HIV-1 containing these mutations showed in viral replication fitness. Whereas the Y501F mutant virus was only in replication the Y501W mutant virus showed a in replication compared with WT and the Y501R virus was to of the essential of Tyr-501 and also suggest that Tyr-501 may be in the of viral fitness. The of antiviral resistance is a major in the of HIV and may be to drugs that essential residues such as Tyr-501. is that HIV-1 resistance to such the of this resistance may be to other thereby Whereas BBNH is one of the few potent inhibitors of HIV-1 RT RNase H so far its potency is significantly other in and as such be as an for the of potent RNase H inhibitors. Our molecular model that the bound BBNH may be of other RT and BBNH that which of these potential in are in

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