Engineering of a Chimeric RB69 DNA Polymerase Sensitive to Drugs Targeting the Cytomegalovirus Enzyme
2009; Elsevier BV; Volume: 284; Issue: 39 Linguagem: Inglês
10.1074/jbc.m109.012500
ISSN1083-351X
AutoresEgor P. Tchesnokov, Aleksandr Obikhod, Raymond F. Schinazi, Matthias Götte,
Tópico(s)Biochemical and Molecular Research
ResumoDetailed structural and biochemical studies with the human cytomegalovirus (HCMV UL54) DNA polymerase are hampered by difficulties to obtain this enzyme in large quantities. The crystal structure of the related RB69 DNA polymerase (gp43) is often used as a model system to explain mechanisms of inhibition of DNA synthesis and drug resistance. However, here we demonstrate that gp43 is ∼400-fold less sensitive to the pyrophosphate analog foscarnet, when compared with UL54. The RB69 enzyme is also able to discriminate against the nucleotide analog inhibitor acyclovir. In contrast, the HCMV polymerase is able to incorporate this compound with similar efficiency as observed with its natural counterpart. In an attempt to identify major determinants for drug activity, we replaced critical regions of the nucleotide-binding site of gp43 with equivalent regions of the HCMV enzyme. We show that chimeric gp43-UL54 enzymes that contain residues of helix N and helix P of UL54 are resensitized against foscarnet and acyclovir. Changing a region of three amino acids of helix N showed the strongest effects, and changes of two segments of three amino acids in helix P further contributed to the reversal of the phenotype. The engineered chimeric enzyme can be produced in large quantities and may therefore be a valuable surrogate system in drug development efforts. This system may likewise be used for detailed structural and biochemical studies on mechanisms associated with drug action and resistance. Detailed structural and biochemical studies with the human cytomegalovirus (HCMV UL54) DNA polymerase are hampered by difficulties to obtain this enzyme in large quantities. The crystal structure of the related RB69 DNA polymerase (gp43) is often used as a model system to explain mechanisms of inhibition of DNA synthesis and drug resistance. However, here we demonstrate that gp43 is ∼400-fold less sensitive to the pyrophosphate analog foscarnet, when compared with UL54. The RB69 enzyme is also able to discriminate against the nucleotide analog inhibitor acyclovir. In contrast, the HCMV polymerase is able to incorporate this compound with similar efficiency as observed with its natural counterpart. In an attempt to identify major determinants for drug activity, we replaced critical regions of the nucleotide-binding site of gp43 with equivalent regions of the HCMV enzyme. We show that chimeric gp43-UL54 enzymes that contain residues of helix N and helix P of UL54 are resensitized against foscarnet and acyclovir. Changing a region of three amino acids of helix N showed the strongest effects, and changes of two segments of three amino acids in helix P further contributed to the reversal of the phenotype. The engineered chimeric enzyme can be produced in large quantities and may therefore be a valuable surrogate system in drug development efforts. This system may likewise be used for detailed structural and biochemical studies on mechanisms associated with drug action and resistance. Infection with the human cytomegalovirus (HCMV), 2The abbreviations used are: HCMVhuman cytomegalovirusUL54catalytic subunit of the HCMV DNA polymeraseCDVcidofovirGCVganciclovirACVacyclovirACV-TPacyclovir triphosphatePFAfoscarnetHSVherpes simplex virusRTreverse transcriptaseHIV-1human immunodeficiency virus, type 1UL30catalytic subunit of the HSV DNA polymerasegp43catalytic subunit of the RB69 DNA polymeraseWTwild typePDBProtein Data Bank. which belongs to the Herpesviridae, remains an important health problem in immunocompromised persons (1Deayton J.R. Prof. Sabin C.A. Johnson M.A. Emery V.C. Wilson P. Griffiths P.D. 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J. Biol. Chem. 1984; 259: 9575-9579Abstract Full Text PDF PubMed Google Scholar). Although active against HCMV, ACV is not approved for treatment of HCMV infection, and its efficacy is inferior to GCV or CDV (8Mercorelli B. Sinigalia E. Loregian A. Palù G. Rev. Med. Virol. 2008; 18: 177-210Crossref PubMed Scopus (65) Google Scholar, 26Talarico C.L. Burnette T.C. Miller W.H. Smith S.L. Davis M.G. Stanat S.C. Ng T.I. He Z. Coen D.M. Roizman B. Biron K.K. Antimicrob. Agents Chemother. 1999; 43: 1941-1946Crossref PubMed Google Scholar, 27Zimmermann A. Michel D. Paviæ I. Hampl W. Lüske A. Neyts J. De Clercq E. Mertens T. Antiviral Res. 1997; 36: 35-42Crossref PubMed Scopus (43) Google Scholar). The pyrophosphate analog foscarnet (phosphonoformic acid, PFA) is the third approved anti-HCMV drug that inhibits UL54 (Fig. 1) (28Oberg B. Pharmacol. Ther. 1989; 40: 213-285Crossref PubMed Scopus (173) Google Scholar, 29Wagstaff A.J. Bryson H.M. Drugs. 1994; 48: 199-226Crossref PubMed Scopus (221) Google Scholar). However, toxicity, problems with oral bioavailability, and the rapid development of resistance can limit the clinical utility of each of the approved drugs. human cytomegalovirus catalytic subunit of the HCMV DNA polymerase cidofovir ganciclovir acyclovir acyclovir triphosphate foscarnet herpes simplex virus reverse transcriptase human immunodeficiency virus, type 1 catalytic subunit of the HSV DNA polymerase catalytic subunit of the RB69 DNA polymerase wild type Protein Data Bank. PFA is a broad spectrum antiviral agent that was shown to inhibit various polymerases, including enzymes encoded by herpes simplex virus (HSV), human herpesvirus, HCMV, and the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) (28Oberg B. Pharmacol. Ther. 1989; 40: 213-285Crossref PubMed Scopus (173) Google Scholar, 29Wagstaff A.J. Bryson H.M. Drugs. 1994; 48: 199-226Crossref PubMed Scopus (221) Google Scholar). Progress has been made in elucidating the mechanism of inhibition of HIV-1 RT (30Marchand B. Tchesnokov E.P. Götte M. J. Biol. Chem. 2007; 282: 3337-3346Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 31Eriksson B.F. Schinazi R.F. Antimicrob. Agents Chemother. 1989; 33: 663-669Crossref PubMed Scopus (71) Google Scholar). Site-specific footprinting experiments revealed that the enzyme can oscillate between two conformations, referred to as pre- and post-translocation (32Marchand B. Götte M. J. Biol. Chem. 2003; 278: 35362-35372Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The 3′ end of the primer still occupies the nucleotide-binding site in the pre-translocated complex (33Boyer P.L. Sarafianos S.G. Arnold E. Hughes S.H. J. Virol. 2001; 75: 4832-4842Crossref PubMed Scopus (241) Google Scholar, 34Götte M. Expert Rev. Anti Infect. Ther. 2004; 2: 707-716Crossref PubMed Scopus (25) Google Scholar). Binding of the next nucleotide requires translocation of the enzyme relative to its nucleic acid substrate (35Götte M. Curr. Pharm. Des. 2006; 12: 1867-1877Crossref PubMed Scopus (29) Google Scholar). The dNTP substrate can bind to and is incorporated in the post-translocated complex. In contrast, PFA traps the pre-translocational complex, which provides a plausible mechanism for inhibition (30Marchand B. Tchesnokov E.P. Götte M. J. Biol. Chem. 2007; 282: 3337-3346Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Marchand B. Götte M. J. Biol. Chem. 2003; 278: 35362-35372Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The mechanism of action might be similar with the HCMV enzyme; however, the limited solubility of UL54 makes it difficult to produce the purified enzyme in sufficient amounts required for detailed biochemical and structural studies (36Ducancelle A. Gravisse J. Alain S. Fillet A.M. Petit F. Pors M.J. Mazeron M.C. J. Virol. Methods. 2005; 125: 145-151Crossref PubMed Scopus (15) Google Scholar, 37Picard-Jean F. Bougie I. Bisaillon M. Biochem. J. 2007; 407: 331-341Crossref PubMed Scopus (13) Google Scholar). Combined in vitro transcription/translation systems and the baculovirus expression system have proven successful for the expression of UL54 and the related HSV polymerase (UL30) (38Loregian A. Rigatti R. Murphy M. Schievano E. Palu G. Marsden H.S. J. Virol. 2003; 77: 8336-8344Crossref PubMed Scopus (49) Google Scholar, 39Loregian A. Appleton B.A. Hogle J.M. Coen D.M. J. Virol. 2004; 78: 158-167Crossref PubMed Scopus (66) Google Scholar, 40Cihlar T. Fuller M.D. Cherrington J.M. J. Virol. 1998; 72: 5927-5936Crossref PubMed Google Scholar, 41Cihlar T. Fuller M.D. Mulato A.S. Cherrington J.M. Virology. 1998; 248: 382-393Crossref PubMed Scopus (75) Google Scholar, 42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar, 43Chaudhuri M. Song L. Parris D.S. J. Biol. Chem. 2003; 278: 8996-9004Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The UL30 apoenzyme has been crystallized (44Liu S. Knafels J.D. Chang J.S. Waszak G.A. Baldwin E.T. Deibel Jr., M.R. Thomsen D.R. Homa F.L. Wells P.A. Tory M.C. Poorman R.A. Gao H. Qiu X. Seddon A.P. J. Biol. Chem. 2006; 281: 18193-18200Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar); however, crystallographic data for UL54 are not available (45Shi R. Azzi A. Gilbert C. Boivin G. Lin S.X. Proteins. 2006; 64: 301-307Crossref PubMed Google Scholar). Like the related phage RB69 DNA polymerase (gp43), UL54 and UL30 belong to the polymerase α family (46Braithwaite D.K. Ito J. Nucleic Acids Res. 1993; 21: 787-802Crossref PubMed Scopus (531) Google Scholar). The RB69 polymerase can be expressed in its soluble form in Escherichia coli, which facilitates protein production at high yields (47Hogg M. Wallace S.S. Doublié S. EMBO J. 2004; 23: 1483-1493Crossref PubMed Scopus (127) Google Scholar, 48Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). This enzyme has also been crystallized in various forms, with and without the bound primer-template and the nucleotide substrate (48Wang J. Sattar A.K. Wang C.C. Karam J.D. Konigsberg W.H. Steitz T.A. Cell. 1997; 89: 1087-1099Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 49Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 50Shamoo Y. Steitz T.A. Cell. 1999; 99: 155-166Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The ternary complex serves as a model system that is often used to explain mechanisms of drug action and resistance associated with UL54 (45Shi R. Azzi A. Gilbert C. Boivin G. Lin S.X. Proteins. 2006; 64: 301-307Crossref PubMed Google Scholar). The structure of the ternary complex shows that helix N and helix P provide important contacts that help to trap the incoming nucleotide (49Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Enzyme kinetic data confirmed the roles played by conserved amino acids in nucleotide binding and catalysis (51Yang G. Lin T. Karam J. Konigsberg W.H. Biochemistry. 1999; 38: 8094-8101Crossref PubMed Scopus (44) Google Scholar, 52Yang G. Franklin M. Li J. Lin T.C. Konigsberg W. Biochemistry. 2002; 41: 2526-2534Crossref PubMed Scopus (51) Google Scholar). Phenotypic drug susceptibility assays suggest that the equivalent regions in UL54 can affect susceptibility to GCV, CDV, and also to PFA (7Gilbert C. Boivin G. Antimicrob. Agents Chemother. 2005; 49: 873-883Crossref PubMed Scopus (179) Google Scholar, 53Baldanti F. Lurain N. Gerna G. Hum. Immunol. 2004; 65: 403-409Crossref PubMed Scopus (68) Google Scholar). Our recent biochemical data have shown that changes at residues of helix P implicated in PFA resistance or hypersusceptibility can either increase or decrease the inhibitory effects of this compound (42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar). A comparison with the crystal structure of gp43 suggests that the binding sites for PFA and the γ-phosphate of the bound nucleotide might overlap. However, although the existence of conserved residues within helix P points to a similar functional role in both enzymes, several residues in this region are different in nature, which may in turn affect sensitivity to PFA (Fig. 2A). Moreover, the contribution of helix N in PFA binding remains to be determined. In light of the limitations with respect to the expression of UL54, and the general shortcomings of surrogate systems such as the RB69 or UL30 enzymes, we engineered a chimeric RB69 DNA polymerase in which critical components of helix N and helix P were replaced by equivalent regions of the related HCMV enzyme (Table 1 and Fig. 2B). The aim of this study was to characterize the roles of the two helices in drug susceptibility to PFA and ACV and, in turn, to improve model systems for more detailed biochemical and structural analyses of UL54 and its interaction with antiviral drugs. The chimeric enzyme retained properties that facilitate expression and purification, and at the same time, the enzyme facilitates the study of mechanisms involved in drug action and drug resistance associated with the clinically relevant HCMV system. Most importantly, we show that gp43 is resistant to PFA and ACV, whereas the chimeric gp43-UL54 enzyme is almost as sensitive to both drugs as seen with UL54.TABLE 1RB69-HCMV DNA polymerase chimeraNomenclature for chimera (block)RegionRB69 residuesHCMV residuesCommentsAHelix NVal-478Trp-780NonePhe-479Val-781Asn-480Ser-782A/V781IHelixChimera A V479IChimera A V781IFoscarnet resistance conferring mutationaA 5.2-fold increase in foscarnet resistance is indicated (62).BHelix PIle-557Met-808NoneAsn-558Ala-809Arg-559Leu-810CHelix PLeu-601Val-812NoneLeu-602Thr-813Ile-603Cys-814ABC/R784AHelix NChimera AChimera AR482AR784AConserved residueHelix PChimera BCChimera BCABC/Q807AHelix NChimera AChimera AHelix PChimera BCChimera BCQ556AQ807AConserved residue conferring resistance to foscarnetbA 6-fold increase in foscarnet resistance is shown (42).a A 5.2-fold increase in foscarnet resistance is indicated (62Baldanti F. Sarasini A. Silini E. Barbi M. Lazzarin A. Biron K.K. Gerna G. Scand. J. Infect. Dis. Suppl. 1995; 99: 103-104PubMed Google Scholar).b A 6-fold increase in foscarnet resistance is shown (42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar). Open table in a new tab Wild-type HCMV polymerase (UL54) was derived from recombinant viruses generated by overlapping cosmids as described previously (40Cihlar T. Fuller M.D. Cherrington J.M. J. Virol. 1998; 72: 5927-5936Crossref PubMed Google Scholar). The UL54 coding sequence was kindly provided by Dr. Guy Boivin (Laval University). The UL54 coding sequence was cloned into pCITE4b (Novagen) by use of the EcoRI and HindIII sites to generate pCITE4b/UL54. We also generated a 3′–5′-exonuclease negative construct that contains the D542A substitution. The RB69 DNA polymerase (gp43) coding sequence was kindly provided by Dr. Sylvie Doublié (University of Vermont) and Dr. Jim Karam (Tulane University). The gp43 coding sequence was cloned into pPR-IBA1 (IBA) using the BsaI site to generate pPR-IBA1/gp43. This construct facilitates protein purification through Strep-tag affinity chromatography (IBA). D222A and D327A substitutions were introduced to remove the 3′–5′-exonuclease activity. Constructs for the production of mutant enzymes were generated by site-directed mutagenesis. The amino acid substitutions were introduced with PfuUltra DNA polymerase (Stratagene) according to the manufacturer's recommendations. The HCMV polymerase UL54 was expressed in rabbit reticulocyte lysate with a coupled in vitro transcription-translation system (Promega). Reactions were conducted essentially as described previously (41Cihlar T. Fuller M.D. Mulato A.S. Cherrington J.M. Virology. 1998; 248: 382-393Crossref PubMed Scopus (75) Google Scholar, 42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar). The RB69 DNA polymerase and chimeric RB69/HCMV enzymes were expressed as described previously (47Hogg M. Wallace S.S. Doublié S. EMBO J. 2004; 23: 1483-1493Crossref PubMed Scopus (127) Google Scholar). All enzymes were purified using Strep-tag affinity chromatography (IBA) according to the manufacturer's recommendations. Heterodimeric reverse transcriptase p66/p51 was expressed and purified as described (54Le Grice S.F. Cameron C.E. Benkovic S.J. Methods Enzymol. 1995; 262: 130-144Crossref PubMed Scopus (121) Google Scholar). Oligodeoxynucleotides used in this study were chemically synthesized and purchased from Invitrogen. The following sequences were used as templates: T1, 5′GTAACTAGAGATCCCTCAGACCCTTTTAGTCAGAAT, and T2, 5′CCAATATTCACCATCAAGGCTTGATGAAACTTCACTCCACTATACCACTC. The underlined nucleotides are the portion of the templates annealed to the primer. The following primers were used in this study: P1, 5′TTCTGACTAAAAGGGTCTGAGGGAT, and P2, 5′GAGTGGTATAGTGGAGTGAA. Deoxynucleotides were purchased from Fermentas Life Sciences, and PFA was purchased from Sigma. ACV (1.5 mmol) was dissolved in 200 μl of dry 1,3-dimethyl-2-oxohexahydropyrimidine, N,N′-dimethylpropylene urea with 12–15 molecular sieves under nitrogen and stirred for 24 h. The mixture was chilled with an ice-water bath and stirred for 1 h, followed by slow addition of phosphorus oxychloride (3 eq) and stirred for an additional 25 min. A solution of tributylammonium pyrophosphate (4 eq) in 200 μl of N,N′-dimethylpropylene and tributyl amine (15 eq) was simultaneously added to the reaction. After 45 min the reaction was quenched with ice-cold water. The reaction was washed with chloroform, and the aqueous layer was collected and co-evaporated with deionized water three times. The residue was resuspended in 100 μl of deionized water and purified on ion-exchange column by high performance liquid chromatography λmax = 253 nm. The final product was co-evaporated with water five times, giving a total yield of ACV-TP (NH3)4 of 18% with purity ≥95%. The molecular weight of the ACV-TP was confirmed by liquid chromatography-mass spectrometry/tandem mass spectrometry m/z (M + 1) 466 → 152 (55Burgess K. Cook D. Chem. Rev. 2000; 100: 2047-2060Crossref PubMed Scopus (194) Google Scholar). 100 nm DNA/DNA primer-template hybrid T1/P1 (100 nm) was preincubated for 5–10 min at 37 °C with a given DNA polymerase in a buffer containing 25 mm Tris-HCl (pH 8), 50 mm NaCl, 0.5 mm dithiothreitol, 0.2 mg/ml bovine serum albumin, and 5% glycerol. To compare different enzymes in single nucleotide incorporation assays, we adjusted the enzyme concentration and the time point of the reaction such that ∼40% of the primer was used at the saturating concentration of dNTP (Fig. 3A). For Km and Ki measurements, the range of the dNTP substrate and/or inhibitor was chosen such that the values were in the middle of the chosen concentration range. Nucleotide incorporation was initiated by the addition of MgCl2 to a final concentration of 10 mm, and the reactions were allowed to proceed for 5 min. The reactions were stopped by the addition of three reaction volumes of formamide containing traces of bromphenol blue and xylene cyanol. Samples were then subjected to 15% denaturing PAGE followed by phosphorimaging. The incorporation of single nucleotides was quantified as the fraction of the DNA substrate (primer n) converted to product (primer n + 1). The rate of the reaction was plotted versus the concentration of nucleotide substrate. The data points of a 42-data point Km/Ki experiment were fit to the general mixed model of inhibition using GraphPad Prism (version 5.0) to calculate kcat, Km, and Ki values. kcat is defined as the enzyme turnover number that is calculated by normalizing the maximum rate of the single nucleotide incorporation reaction to the enzyme concentration. Km is defined as the dNTP substrate concentration at half-maximum rate of the reaction. Ki is the inhibitor dissociation constant (56Copeland R.A. Evaluation of Enzyme Inhibitors in Drug Discovery: A Primer for Medicinal Chemists and Pharmacologists. John Wiley & Sons Inc., Hoboken, NJ2005: 34-37Google Scholar, 57Copeland R.A. Enzymes. 2nd Ed. John Wiley & Sons Inc., New York2000: 109-145Crossref Google Scholar). Significant figures for the fitted data of all experiments are as reported by the software. Standard deviations for all experiments were determined on the basis of at least three independent replicates. The incorporation of dCTP was determined by plotting the percentage of incorporation against the concentration of PFA (Fig. 3). IC50 values were calculated by fitting at least 10 data points to a sigmoidal dose-response (variable slope) equation using GraphPad Prism (version 5.0). Significant figures for the fitted data of all experiments are as reported by the software. Standard deviations for all experiments were determined on the basis of at least three independent replicates. The secondary structure prediction-based alignment between UL54 and UL30 (PDB code 2GV9) was generated through ESyPred3D Web Server 1.0 (58Lambert C. Léonard N. De Bolle X. Depiereux E. Bioinformatics. 2002; 18: 1250-1256Crossref PubMed Scopus (529) Google Scholar). The result was uploaded into the crystal structure-based alignment between UL30 (PDB code 2GV9) and gp43 (PDB code 1IG9 (49Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar)) using the chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (59Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (29006) Google Scholar) The sequence alignment output was graphically prepared with ESPRIPT software (60Gouet P. Robert X. Courcelle E. Nucleic Acids Res. 2003; 31: 3320-3323Crossref PubMed Scopus (1107) Google Scholar). Conserved residues are highlighted in black, and similar residues are boxed (61Gouet P. Courcelle E. Stuart D.I. Métoz F. Bioinformatics. 1999; 15: 305-308Crossref PubMed Scopus (2540) Google Scholar). The goal of this study was to engineer and to characterize a chimeric RB69/UL54 enzyme that facilitates the study of PFA-mediated inhibition of DNA synthesis and drug resistance. We focused on segments of helix N and helix P that are located in close proximity to the phosphates of the bound dNTP substrate in the ternary complex with gp43 (Table 1 and Fig. 2) (49Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). Several amino acids in these regions are likewise implicated in binding of PFA by UL54. Phenotypic susceptibility assays, and our previous biochemical data suggest that the region between 807 and 815 in helix P (556–564 in gp43) plays an important role in this regard (7Gilbert C. Boivin G. Antimicrob. Agents Chemother. 2005; 49: 873-883Crossref PubMed Scopus (179) Google Scholar, 42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar). Some of the amino acids of this segment can interact with the bound nucleotide, whereas others appear to be involved in interhelical interaction with residues 779–784 (477–482 in gp43) of helix N (49Franklin M.C. Wang J. Steitz T.A. Cell. 2001; 105: 657-667Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar, 50Shamoo Y. Steitz T.A. Cell. 1999; 99: 155-166Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) (Fig. 2B). In an attempt to closely mimic the structure of UL54, we replaced amino acids that differ at equivalent positions in gp43 according to the sequence alignment shown in Fig. 2A. These changes involve the block of three amino acids between the conserved residues Lys-477 and Gln-481/Lys-482 of helix N, referred to as block A, and two blocks of three amino acids between conserved residues Gln-556 and Asn-564 of helix P, referred to as block B and block C, respectively. Of note, the alignment also points to several amino acid changes in equivalent blocks A, B, and C of UL54 and UL30, respectively. All enzymes designed and characterized in this study are listed in Table 1. We generated chimeric gp43-based enzymes in which block A of helix N and blocks B/C of helix P have been replaced by equivalent regions of UL54. Several conserved amino acids within these regions interact with the phosphates of the nucleotide (Fig. 2B). Arg-482 of helix N as well as Gln-556 of helix P appear to contact the γ-phosphate of the bound nucleotide. The same region is also implicated in PFA binding. The Q807A mutation in helix P in UL54 increases the IC50 value for PFA (42Tchesnokov E.P. Gilbert C. Boivin G. Götte M. J. Virol. 2006; 80: 1440-1450Crossref PubMed Scopus (27) Google Scholar). However, a potential role of helix N in PFA resistance remains to be defined. Of note, Phe-479 of helix N in gp43 is equivalent to Val-781 in UL54, and the V781I substitution in the HCMV enzyme shows decreased phenotypic susceptibility to PFA (62Baldanti F. Sarasini A. Silini E. Barbi M. Lazzarin A. Biron K.K. Gerna G. Scand. J. Infect. Dis. Suppl. 1995; 99: 103-104PubMed Google Scholar). This residue is likewise located in the vicinity of the γ-phosphate, and this region appears to be important for PFA binding. Thus, to test how close the chimeric enzyme may mimic the natural HCMV polymerase, we introduced V781I and alanine substitutions at conserved residues Arg-784 (helix N) and Gln-807 (helix P), respectively, against the background of the chimera. We initially determined steady-state kinetic parameters for single nucleotide incorporation events, and we compared several chimeric enzymes with WT gp43 (Table 2). Throughout this study, we used 3′–5′-exonuclease negative mutants to prevent potentially confounding effects through the editing activity. Replacing block A (helix N) with the equivalent UL54 region caused ∼2–3-fold reductions in the efficiency of single nucleotide in
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