Fidelity of hepatitis B virus polymerase Sung Gyoo Park 1 , Younhee Kim 2 , Esther Park 1 , Hyun Mi Ryu 1 and Guhung Jung 1 1 School of Biological Science, Seoul National University, Seoul; 2 Department of Oriental Medicine, Semyung University, Checheon, Chungbuk, Korea Although efficient vaccines are available, chronic hepatitis B (HBV) infection poses a major health problem worldwide, and prolonged treatment of chronically infected HBV patients with nucleoside analogs often results in drug- resistant HBV variants.Therefore, it is critical to evaluate the contribution of the HBV polymerase to mutations. FLAG- tagged wild-type (FPolE) and mutant (FPolE/D551A) HBV polymerases have been expressed in insect cells and purified. The purified FPolE showed DNA polymerase activity, but FPolE/D551A did not, implying that the activity was derived from FPolE. No 3¢fi5¢ exonuclease activity was detected in FPolE. The fidelity of FPolE was investigated and compared with that of HIV-1 RT, which is highly error- prone. The fidelity of HBV polymerase seems to be achieved by increasing the K m for the dNTP being misinserted. The nucleotide misinsertion efficiency of FPolE and HIV-1 RT ranged from 3.59 · 10 )4 (C : T) to 1.51 · 10 )3 (G : T) and from 1.75 · 10 )4 (C : T) to 1.62 · 10 )3 (G : T), respect- ively, and the overall misinsertion efficiency of HIV-1 RT was just 1.04-fold higher than that of FPolE, implying that HBV polymerase is fairly error-prone. Though HBV genetic mutation rate in replication is thought to be between those in RNA and DNA viruses, our data shows that the rate of mutation by HBV polymerase is higher than the rate of genetic mutation in vivo. This may be a result from more overlapping HBV genes in the HBV genome than that of other retroviruses. Keywords: HBV polymerase; HBV; fidelity; misinsertion; mispair; exonuclease. The hepatitis B virus (HBV) is a member of the hepadnavi- ridae, a family of enveloped hepatotropic DNA viruses. The virus can cause severe liver disease with eventual progression to cirrhosis and primary hepatocellular carcinoma. Never- theless, the number of chronic HBV carriers is estimated to exceed 350 million [1] and HBV chronic infection remains among the 10 most common causes of death worldwide according to the 1997 World Health Organization report [2]. Moreover, deaths from liver cancer caused by HBV infection probably exceed one million per year worldwide [3,4]. The mature virus consists of a partially duplex, relaxed circular genome of 3.2 kb [5,6]. The HBV genome contains four open reading frames (ORFs) coding for the viral core antigen, the viral surface antigen, the viral DNA polym- erase, and the transactivator protein X. Unlike most DNA viruses, it replicates via reverse transcription of an RNA intermediate and is distantly related to the retroviruses. The process includes polymerization of minus-strand DNA (by RNA-dependent DNA polymerization), degradation of the pregenome from an RNA-DNA heteroduplex (by RNase H activity) as minus-strand DNA synthesis proceeds, and synthesis of plus-strand DNA from the minus-strand DNA template (by DNA-dependent DNA polymerization). All enzyme activities responsible for these steps come from the viral polymerase. Mutational and sequence analyses among the coding region of P ORF and those of several retroviral reverse transcriptases (RTs) revealed that they have sequence homology [7,8] and that there are four domains within the P ORF: a terminal protein at the N-terminus followed by a spacer region, a reverse transcriptase, and a C-terminal RNase H domain [9–11]. Retroviruses exhibit a relatively high rate of mutation attributed to the inaccuracy of the replication machinery that is unique to the retroviral life cycle [12]. The generation of HIV variants is facilitated by the overall low polymerase fidelity of viral reverse transcriptase [13– 15]. As RT is a preferred target for the development of viral inhibitors as antiviral drugs, researches have focused on the structural and catalytic properties of RTs, inclu- ding three-dimensional crystal studies [16–18]. Because HBV RT is functionally and structurally related to HIV RT, some of the nucleoside analogs (such as lamivudine) developed to treat HIV infection are highly potent against HBV infection [1,19] at concentrations below cytotoxic thresholds [20]. However, short-term monotherapy with lamivudine is insufficient to clear viral infection and prolonged use has caused the increased emergence of lamivudine-resistant HBV [21]. HBV polymerase mutants may occur due to the fast viral turnover rate [22], which may lead to the heterogeneity of HBV viral genomes. Mutations of viral genomes also result in the existence of quasispecies in infected individ- uals that evolve during the course of infection depending on the host selective pressure [23]. The existence of HBV as quasispecies may be favored by the infidelity of HBV Correspondence to G. Jung, School of Biological Sciences, Seoul National University, Seoul, 151–742, Korea. Fax: + 82 2 8807773, Tel.: + 82 2 8807773, E-mail: drjung@snu.ac.kr Abbreviations: AMV, avian myeloblastosis virus; HBV, hepatitis B virus; FPolE, FLAG-fused HBV polymerase; MLV, murine leukemia virus; ORF, open reading frame; PVDF, poly(vinylidene difluoride); RT, reverse transcriptase. (Received 24 March 2003, accepted 2 May 2003) Eur. J. Biochem. 270, 2929–2936 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03650.x polymerase, which would account for the emergence of the many natural mutants with point substitutions and the hypermutation phenomenon [24]. Naturally occurring mutations of HBV have been identified in all the HBV viral genes and regulatory elements, indicating that HBV mutation may affect infection, viral clearance, and response to antiviral therapy [25]. While research toward understanding the extent and source of HBV variation is lacking, fidelity of HIV-1 RT has been investigated by many researchers in in vitro conditions using Pol expressed in heterologous systems. Thus, it is important to evaluate the contribution of the HBV polymerase to mutations. In this study, FLAG-tagged wild-type and catalytic mutant HBV polymerases have been expressed in insect cells and purified using immunoaffinity column chromatogra- phy. We show that the purified HBV polymerase exhibits DNA-dependent DNA polymerase activity, but the purified mutant HBV polymerase does not show the activity. This result indicates that polymerase activity is not caused by host polymerase contamination. In addition, the purified wild-type polymerase does not have 3¢fi5¢ exonucleolytic proofreading activity, like other RTs. The nucleotide insertion fidelity of the HBV polymerase was examined and compared with that of HIV-1 RT, and the result shows that HBV polymerase may have similar mutation rates to HIV-1 RT. This is the first study on the fidelity of HBV polymerase. Experimental procedures Materials T4 polynucleotide kinase was purchased from New England Biolabs. Unlabeled nucleotides were purchased from Phar- macia. Oligonucleotides were synthesized by Integrated DNA Technology Inc. Homopolymer template poly(dA)¢- oligo(dT) 12)18 was obtained from Amersham Pharmacia, and [a- 32 P]dTTP (3000 CiÆmmol )1 ) was purchased from NEN Life Science Products. HIV-1 RT (specific acti- vity, > 5000 UÆmg )1 ) was purchased from Roche Mole- cular Biochemicals. Poly(vinylidene difluoride) (PVDF) blotting membrane was from Millipore, and M2 mono- clonal antibody was from Sigma. Methods Construction and purification of the wild-type and mutant HBV polymerase. Two recombinant plasmids, pFPolE and pFPolE/D551A, containing entire HBV polymerase gene (subtype adr [26]), and catalytic mutant HBV poly- merase gene, respectively, with FLAG sequences at the N-terminal region were used. The FLAG tag was used to isolate the wild-type and mutant HBV polymerase. pFPolE/ D551A has a mutation in nucleotide 1654 changing A to C, altering amino acid residue 551 from aspartic acid to alanine. Each recombinant baculovirus was expressed in Sf-9 cells, and the proteins were purified as described previously [27]. SDS/PAGE and immunoblot analysis. The partially puri- fied proteins were separated by 7.5% SDS/PAGE. For immunoblot analysis, proteins were electrophoretically transferred to a PVDF blotting membrane. The membranes were probed with M2 monoclonal antibody and resus- pended in NaCl/P i containing 0.5% skimmed milk and 0.3% Tween-20. The immunoblots were then incubated with horseradish peroxidase-conjugated antimouse anti- serum. The immunoreactive bands were visualized using the ECL system (Amersham Pharmacia). DNA polymerase activity. The DNA polymerization reaction (total reaction volume of 50 lL) contained 50 ng of homopolymer template poly(dA)¢oligo(dT) 12)18 , 2 lCi of [a- 32 P]dTTP (3000 CiÆmmol )1 ), 50 m M Tris/HCl pH 7.4, 0.01% NP-40, 10 m M MgCl 2 ,1m M dithiothrei- tol, 10 m M KCl and 50 l M unlabeled dTTP. The reaction was started by adding 90 ng (1 pmol) of either the purified proteins of wild type (FPolE) or mutant (FPolE/ D551A), and incubated at 37 °C for 30 min. The reaction was quenched by the addition of 2 lL0.5 M EDTA, and the reaction products were phenol-extracted and ethanol- precipitated. Two microliters of 95% formamide were added and the proteins were immediately denatured by incubating at 95 °C for 3 min and analyzed by electro- phoresis in 7 M urea/16% polyacrylamide gels. The gel was then dried and exposed to a phosphoimager system (BAS FLA2000, Japan). Template-primers. Different template-primer substrates were used for measuring exonuclease activity and site- specific nucleotide misinsertion (Table 1). Each primer was end-labeled with [c- 32 P]ATP (3000 CiÆmmol )1 ), using T4 polynucleotide kinase (20 U). Reaction was started by adding 100 n M oligonucleotide and incubated at 37 °Cfor 1.5 h, and stopped by adding EDTA to the final concen- tration of 20 m M . The reaction mixture was then phenol- extracted twice and ethanol-precipitated. To measure the 3¢fi5¢ exonuclease activity, hybrid molecules between 16-mer oligonucleotide and 24-mer template were made. To measure site-specific nucleotide misinsertion, four different primers (1510G, 2226A, 5385T and 1212C) were hybridized to the M13mp18 single-stranded template. Partially double- stranded template-primer structures were created by Table 1. Template/primers used in exonuclease activity assay and site- specific misinsertion assay. Exonuclease activity assay Primer 5¢-CCC CTA GAA GAA GAA G 3¢ Template 3¢-GGG GAT CTT CTT CTT AGG ATA GCG-5¢ Site-specific misinsertion assay Primer 1510 G: 5¢-GTT TAT CAG CTT GCT TT- M13mp18: -CAA ATA GTC GAA CGA AAG- Primer 2226 A: 5¢-TGA TAT TCA CAA ACG AA- M13mp18: -ACT ATA AGT GTT TGC TTA- Primer 5385T: 5¢-TTT TAG ACA GGA ACG GT- M13mp18: -AAA ATC TGT CCT TGC CAT- Primer 1212C: 5¢-GTT TTC CCA GTC ACG AC- M13mp18: -CAA AAG GGT CAG TGC TGC- 2930 S. G. Park et al. (Eur. J. Biochem. 270) Ó FEBS 2003 combining 740 n M 32 P-end-labeled primer with 4 l M tem- plate in 50 m M Tris/HCl pH 7.4, 5 m M MgCl 2 ,2m M 2-mercaptoethanol, and 17 lg bovine serum albumin. The mixture was heated at 95 °C and allowed to cool down slowly to room temperature. Exonuclease activity. The 3¢fi5¢ exonuclease activity was measured by the removal rate of mismatched 3¢-terminal nucleotides from the 5¢-[c- 32 P] end-labeled oligonucleotide. The reactions were carried out 25 lL reaction mixture containing 300 ng mismatched template-primer, 50 m M Tris/HCl pH 7.4, 10 m M KCl, 10 m M MgCl 2 ,1m M dithiothreitol, and 0.01% NP-40. The reaction was started by adding 1 pmol FPolE, 100 mU Klenow fragment of Escherichia coli polymerase I as a positive control, or 10 mU HIV-1 RT as a negative control. After incubating for 30 min at 37 °C, the reactions were stopped by adding equal volumes of formamide dye mix. The reaction mixtures were electrophoresed in 7 M urea/16% polyacrylamide sequen- cing gels, and dried. The dried gel was exposed to the phosphoimager system. Site-specific nucleotide misinsertion. The template-primer substrates for measuring the rates of dNTP incorporation opposite the G, A, T and C are shown in Table 1. Before measuring the kinetic constants of correct and wrong nucleotide incorporation, a time course study was carried out to decide the time frame during which products accumulated linearly with time and less than 30% of the original primer was extended [28]. Reaction times were chosen to be 30 min for FPolE, 1 min for correct insertion and 4 min for misinsertion for HIV-1 RT according to the results of time course experiments (data not shown). The specific activity of the partially purified enzyme was 40 unitsÆlg )1 . One unit is defined as the amount of enzyme that catalyzes the incorporation of 1 pmol of dTTP into DNA in the poly(dA) n Æoligo(dT) 12)18 -directed reaction in 30 min at 37 °C. Reactions were started by combining 4 lLenzyme- primer-template solution (12.5 n M FPolE or 5.3 n M HIV-1 RT, 25 lg bovine serum albumin and 2 lL of the original annealed primer-template solution), 4 lL dNTP-salts solu- tion (52 m M Tris/HCl pH 7.8, 20 m M MgCl 2 ,5m M dithiothreitol, 150 lg bovine serum albumin and increasing concentrations of single dNTP), and were incubated at 37 °C. The reaction was terminated by the addition of EDTA to a final concentration of 50 m M in 95% form- amide buffer. Reaction products were denatured by incu- bating at 95 °C for 3 min and analyzed by electrophoresis in 16% polyacrylamide/7 M urea gels. Analysis of deoxy- nucleotide incorporation assays was carried out using a gel based steady-state kinetic assay [28,29] to determine misinsertion efficiency for all the mispairs. Gel band intensities of the substrates and products were quantitated using the phosphoimager system within the linear response range. For each concentration of dNTP, the observed rate of deoxynucleotide incorporation (V obs )wasdeterminedby dividing the relative amount of the extended product by the incubation time. The observed rate of deoxynucleotide incorporation was plotted as a function of dNTP concen- tration, and the data were fitted to the Michaelis–Menten equation using nonlinear least-squares methods. Apparent K m and V max steady-state parameters for the incorporation of the correct and incorrect deoxynucleotides were obtained from the fit and used to calculate the frequency of nucleotide misinsertion (f ins )[30]. Results Expression and purification of FLAG-fused wild-type (FPolE) and mutant (FPolE/D551A) HBV polymerase in insect cells The reverse transcriptase domain of HBV polymerase has several conserved motifs, and the YMDD motif is involved in nucleotide binding in the catalytic site of the polymerase [31,32]. In this study, to determine whether polymerase activity of purified wild-type HBV polymerase due to contamination by host polymerase or not, single amino acid change was made in this YMDD motif and the mutant is called FPolE/D551A. FLAG-fused wild-type and mutant HBV polymerases were expressed in insect cells using the recombinant baculovirus expression system. Sf-9 cells were infected with the FPolE or FPolE/D551A baculovirus and were harvested 48 h postinfection. From the infected cells, the polymerases were purified as described in the Materials and methods. In the purified fractions of FPolE (lane 1) and FPolE/D551A (lane 2), five prominent proteins with a molecular weight of approximately 110, 84, 70 and 60 kDa were eluted as shown in Fig. 1A. The purified 84 kDa protein was immuno-stained with the M2 monoclonal antibody that is specific to the FLAG epitope, representing the band was recombinant FPolE or FPolE/D551A proteins as shown in Fig. 1B. In addition through MAL- DI-TOF analysis, it has been reconfirmed that the 84 kDa Fig. 1. Expression and purification of FLAG-tagged wild-type (FPolE) and mutant (FPolE/D551A) HBV polymerases. Sf-9 cells were infected with the FLAG-tagged wild-type (vFPolE) or mutant (vFPolE/ D551A) baculovirus, and were harvested 48 h postinfection. FPolE (lane 1) and FPolE/D551A (lane 2) proteins were partially purified with an affinity resin containing the M2 monoclonal antibody, separ- ated by 7.5% SDS/PAGE, and stained with Coomassie blue (A). For immunoblot analysis, proteins were electrophoretically transferred to a PVDF blotting membrane (Millipore), probed with M2 monoclonal antibody (Sigma), and the immunoblots were then incubated with horseradish peroxidase-conjugated anti-mouse serum (B). Lane 1, molecular mass standards are indicated in kDa. The arrows indicate the position of FPolE and FPolE/D551A on the right. Ó FEBS 2003 Fidelity of HBV polymerase (Eur. J. Biochem. 270) 2931 protein is the two HBV polymerases (FPolE and FPolE/ D551A). Possibly, 110 kDa protein is endogenous protein that binds both tightly and nonspecifically to M2 agarose resin. The other two proteins (70 kDa and 60 kDa proteins) were Hsp70 and Hsp60, respectively. The function of the Hsps for HBV polymerase is described in previous studies [27,33]. DNA polymerase activity The purified HBV polymerase showed DNA-dependent DNA polymerase activity (Fig. 2, lane 1). To confirm that the polymerase activity is from HBV polymerase, poly- merase assays were performed with the purified FPolE/ D551A which lacks polymerase activity due to a point mutation. Under the standard reaction conditions described in Experimental procedures, polymerase reactions were conducted with the purified fractions of wild-type or mutant HBV polymerase. Reaction products from FPolE or FPolE/D551A were subjected to electrophoresis in a 7 M urea/16% polyacrylamide sequencing gel as shown in Fig. 2. Polymerization products were detected from FPolE (lane 1), but not from FPolE/D551A (lane 2), indicating that the polymerization activity was clearly derived from the FPolE. Analysis of the 3¢fi fi 5¢ exonuclease activity All the RTs studied thus far lack 3¢fi5¢ exonuclease activity [34]. Therefore, it was interesting to check whether the polymerase of this small DNA virus, HBV, displaying similarities to retroviral transcriptases, has any exonuc- lease activity. Terminal mismatched template-primer pairs (Table 1) were incubated with the partially purified FPolE proteins (Fig. 3). Terminal nucleotide excision capability was analyzed in the presence of no proteins (lane 1), HIV-1 RT (lane 2), FPolE proteins (lane 3) and the Klenow fragment of E. coli polymerase I (lane 4). No 3¢fi5¢ exonuclease activity was found in HIV-1 RT (lane 2). There was also no change in the length of the oligonucleotide primer when the FPolE proteins were used (lane 3), in the same way as with HIV-1. However, efficient excision of the terminal nucleotide was found to occur when the Klenow fragment of E. coli polymerase I was used as a positive control (lane 4). Thus, HBV polymerase does not have 3¢fi5¢ exonuclease activity, as isthecasewithmanyRTs. Site-specific nucleotide misincorporation The ratio of the insertion efficiency for wrong (W) vs. right (R) base pairs indicates frequency of nucleotide misinsertion, f ins . The nucleotide insertion fidelity is defined as the reciprocal of f ins : f ins ¼ V max /K m ) W /(V max / K m ) R [17]. The lack of proofreading activity permits the sole analysis of the fidelity of DNA polymerization activity. To determine the nucleotide misinsertion fre- quency of FPolE and HIV-1 RT, we measured the V max and K m steady-state parameters for the incorporation of correct and incorrect deoxynucleotides (G, A, T and C) opposite the G, A, T and C residues on native M13mp18 template-strand primed with 5¢- 32 P end-labeled oligo- nucleotide primers 1510G, 2226A, 5385T and 1212C (Table 1). Four separate reactions were carried out and Fig. 2. Polymerase activity assay. Immunoaffinity-purified wild type (FPolE) and the catalytic site mutant (FPolE/D551A) HBV poly- merases were assayed for polymerizing activity as described in Experimental procedures. Reaction products were subjected to 16% polyacrylamide/urea gel and the dried gel was exposed to the phos- phoimager system. FPolE showed polymerization activity (lane 1), whereas FPolE/D551A (lane 2) did not. Fig. 3. Electrophoretic analysis of terminal mismatch excision. Reac- tions for terminal mismatched (G : A) excision were performed as described in Experimental procedures with no enzyme (lane 1), HIV-1 RT (lane 2), FPolE (lane 3) and Klenow fragment of E. coli poly- merase I (lane 4). The position of the 16-mer primer is indicated by an arrow. The direction of the electrophoresis is from top to bottom. 2932 S. G. Park et al. (Eur. J. Biochem. 270) Ó FEBS 2003 each reaction included a single dNTP to measure the rate of synthesis of the correct pair and three possible mispairs. From the quantitation of unextended and extended primers from each reaction set, the initial velocities of product formation were plotted against the dNTP concentrations, and the double-reciprocal plots for the initial velocities vs. the substrate concentrations were made. Only the data of primer 5385T-template are shown to avoid overlapping of data (Fig. 4). The f ins values for each of the 16 possible insertion events, i.e. four correct Fig. 4. Kinetic assay for site-specific nucleotide misinsertion. The 5¢-[ 32 P] labeled primer 1510G, 2226 A, 5385T or 1212C was annealed to M13mp18 template strand to produce the 3¢-terminal mispairs in the presence of increasing concentrations of single dNTP as indicated, with FPolE or HIV-1 RT. The data pertaining only to the primer 5385T-template are shown. Ó FEBS 2003 Fidelity of HBV polymerase (Eur. J. Biochem. 270) 2933 base pairs and 12 mispairs, were then derived from the apparent K m and V max kinetic values for each dNTP calculated from the double-reciprocal plots, and they were summarized in Table 2. The f ins data from Table 2 were plotted against each mispair (Fig. 5). The range of f ins values of HBV polymerase for all the mispairs was from 3.59 · 10 )4 to 1.51 · 10 )3 with the average f ins value of 6.28 · 10 )4 (1/1591), whereas that by HIV-1 RT was from 1.75 · 10 )4 to 1.62 · 10 )3 with the average f ins value of 6.03 · 10 )4 (1/1658). These figures show that HBV polymerase may also be error-prone, considering that HIV-1 RT is a highly error-prone enzyme. As it is evident from the f ins value analysis, HBV polymerase displayed a rather higher insertion fidelity in purine–purine, purine– pyrimidine and pyrimidine–purine mispairs, but lower insertion fidelity in pyrimidine–pyrimidine mispairs, com- pared with HIV-1 RT (Fig. 5). Conclusively, an average misinsertion efficiency of HIV-1 RT was 1.04-fold higher than that of HBV polymerase. Discussion HBV has the smallest genome of all known human DNA viruses (3.2 kb) and a unique replication strategy with a reverse transcription step. In retroviruses, reverse transcrip- tion is error-prone, which contributes to the high gen- etic variability of retroviruses with the mutation rates of 10 -4 )10 -5 misincorporation per base [13,35]. In wild-type isolates of HBV, the sequence of the genome may vary up to 10% despite conservation of open reading frame and function [36]. Published HBV genomes showed high nuc- leotide sequence variability in S, C and P genes, region X, the precore region, and the pre-S2/pre-S1 regions ranked in the order of increasing variability [37]. HBV mutants affecting all known reading frames of the viral genome have been demonstrated in patients with fulminant or chronic HBV infection. Moreover, novel variants of HBV genomic sequences from patients with unusual serological profiles are continually discovered. The exact contribution of the mutations to the natural course of HBV infection remains to be elucidated, but the genetic variations of HBV are possibly related to the infidelity of the HBV polymerase and reverse transcription strategy of HBV. Fidelity of DNA synthesis is a major determinant in generating spontaneous mutation. However, the molecular mechanisms governing fidelity of DNA synthesis are largely unknown. Judging from the spontaneous mutation rates, the frequency of errors during DNA replication in pro- karyotic and eukaryotic cells are between 10 )9 and 10 )10 substitutions per base pair in each cell generation [38]. These low mutation rates are achieved by multiple steps in error discrimination including base selection by DNA poly- merase, 3¢fi5¢ exonucleolytic proofreading, and post- replicative repair [39]. In the present study, HBV polymerase was found to lack 3¢fi5¢ proofreading exon- uclease activity like all RTs studied so far, suggesting that it Fig. 5. Relative misinsertion efficiencies (f ins ) by HBV polymerase (FPolE) and HIV-1 RT. A comparative plot of misinsertion efficiencies, f ins , for individual mispairs from Table 2 are given in bar graph form. Table 2. The apparent V max , K m values and misinsertion frequency (f ins ) for wild-type HBV polymerase (FPolE) and HIV-1 RT. Data shown are the mean values ± standard deviation. Standard deviations presented are derived from three (FPolE) or two (HIV-1 RT) independent measurements and the variations were mostly <20%. Misinsertion frequency, f ins , were evaluated from ratio of relative V max to K m as using the equation f ins ¼ (V max /K m ) correct /(V max /K m ) incorrect . Base pairs are shown with the template (T) first. Base pair (T:dNTP) K m (l M ) V max (% min )1 ) f ins 1/f ins FPolE HIV-1 RT FPolE HIV-1 RT FPolE HIV-1 RT FPolE HIV-1 RT G : G 61.9 ± 4.5 28.4 ± 2.4 0.161 ± 0.011 4.40 ± 0.81 7.05 · 10 )4 1.61 · 10 )3 1418 618 G : A 49.3 ± 3.7 46.6 ± 5.1 0.195 ± 0.001 5.91 ± 1.35 1.07 · 10 )3 1.32 · 10 )3 932 756 G : T 43.8 ± 5.4 42.0 ± 4.5 0.244 ± 0.036 6.53 ± 0.33 1.51 · 10 )3 1.62 · 10 )3 662 616 G : C 0.0900 ± 0.0020 0.0936 ± 0.0003 0.332 ± 0.035 8.97 ± 0.88 1 1 1 1 A : G 31.1 ± 3.3 31.4 ± 0.1 0.290 ± 0.006 8.67 ± 1.77 1.19 · 10 )3 1.18 · 10 )3 838 842 A : A 45.0 ± 5.2 25.2 ± 2.3 0.192 ± 0.023 7.85 ± 1.88 5.45 · 10 )4 1.33 · 10 )3 1833 746 A : T 0.0404 ± 0.0088 0.0486 ± 0.010 0.316 ± 0.036 11.3 ± 3.8 1 1 1 1 A : C 33.1 ± 1.4 20.0 ± 0.2 0.269 ± 0.021 6.26 + 0.11 1.03 · 10 )3 1.34 · 10 )3 962 742 T : G 202 ± 20 32.9 ± 1.3 0.606 ± 0.095 20.1 ± 0.7 4.88 · 10 )4 1.57 · 10 )3 2046 633 T : A 0.122 ± 0.011 0.0674 ± 0.0033 0.749 ± 0.033 26.1 ± 4.1 1 1 1 1 T : T 98.4 ± 7.6 71.8 ± 9.8 0.357 ± 0.054 15.7 ± 0.4 5.90 · 10 )4 5.64 · 10 )4 1692 1770 T : C 96.5 ± 10.9 39.9 ± 3.0 0.391 ± 0.039 9.58 ± 0.14 6.59 · 10 )4 6.20 · 10 )4 1515 1612 C : G 0.0470 ± 0.0035 0.0676 ± 0.012 0.655 ± 0.086 46.4 ± 6.0 1 1 1 1 C : A 94.7 ± 4.1 76.9 ± 5.5 0.541 ± 0.059 23.3 ± 4.1 4.09 · 10 )4 4.41 · 10 )4 2439 2265 C : T 58.1 ± 3.5 108 ± 2 0.291 ± 0.014 13.0 ± 2.0 3.59 · 10 )4 1.75 · 10 )4 2782 5702 C : C 36.8 ± 3.4 45.2 ± 5.7 0.260 ± 0.041 8.64 ± 0.95 5.06 · 10 )4 2.78 · 10 )4 1972 3590 2934 S. G. Park et al. (Eur. J. Biochem. 270) Ó FEBS 2003 has high mutation rate, at least during DNA replication. Especially for RT lacking proofreading activity, nucleotide misinsertion rates are important parameters contributing to the overall polymerase fidelity [39], but it is not the only factor because retroviral RTs lacking a proofreading exonuclease, such as avian myeloblastosis virus (AMV) and murine leukemia virus (MLV) RT have 10-fold and 18-fold higher fidelity than HIV-1 RT, respectively [35]. Because HIV-1 RT is a well-studied enzyme, there are many reports about the error rate of the enzyme. Misinser- tion efficiency of HIV-1 RT for all the possible mispairs were between 5.60 · 10 )5 (C : T) and 1.55 · 10 )2 (G : T) [40], and misinsertion efficiency for some mispairs were 4.4 · 10 )5 (C : T), 1.2 · 10 )4 (T : T) and 1.6 · 10 )4 (G : T) on oligonucleotide DNA template, and 1.2 · 10 )4 (C : T), 1.8 · 10 )4 (T : T) and 4.4 · 10 )4 (G : T) on M13 DNA template [15]. Another report shows that f ins values of HIV-1 RT for some mispairs were 1/6000 (1.7 · 10 )4 ) (A : C), 1/32 550 (3.1 · 10 )5 ) (A : G), and 1/75 000 (1.3 · 10 )5 ) (A : A) [41]. In contrast, our f ins data for HIV-1 RT were from 1.75 · 10 )4 (1/5714) to 1.62 · 10 )3 (1/6172) with the overall f ins value for all the possible mispairs of 6.03 · 10 )4 (1/1658). Although reported f ins values for HIV-1 RT are variable depending on template and assay system, our data are within the reported range, representing the reliability of our data. In this report, overall misinsertion efficiency for HBV polymerase was 6.28 · 10 )4 (1/1591), and the error rates for each G : T, A : C and A : A mismatch were 1.51 · 10 )3 ,1.03· 10 )3 and 5.45 · 10 )4 , respectively (Table 2). Conclusively, HBV polymerase is fairly error- prone, compared to other reported RTs for misincorpora- tion of nucleotides on the DNA templates. The fidelity of DNA synthesis by HIV-1 RT is several- fold higher with an RNA template than with a DNA template. Misaligned intermediates are formed less fre- quently with an RNA template than with a DNA template [42]. However, there are some reports that the parameters for fidelity of DNA synthesis in vitro depend primarily on the sequences of nucleic acids copied, rather than DNA or RNA templates [34]. In addition to efficient misinsertion, efficient extension of mismatched 3¢-termini of the nascent DNA was found to be a major factor for the infidelity of HIV-1 and HIV-2 RTs [43,44]. Therefore, it is important to examine insertion and extension efficiency on both RNA- and DNA-templated DNA synthesis reactions by HBV DNA polymerase, given the possible role of replication infidelity in generating mutant viruses. As the present study only focuses on the fidelity of misinsertion of nucleotides on the DNA template, a more extensive study remains to be performed to reveal the relationships between fidelity of HBV polymerase and genetic variability. In this report, HBV polymerase is shown to be highly error-prone, compared to other reported RTs, in contrast to a previous report that HBV and the related animal hepadnaviruses are known to have a mutation rate which is intermediate between DNA and RNA viruses [45]. Although HBV polymerase shows a high error rate similar to HIV-1 reverse transcriptase in in vitro conditions, mutation rate of HBV is lower compared to that of HIV-1 in in vivo conditions. The reason may be due to the fact that mutations in HBV are not well tolerated because of more overlapping reading frames in HBV than other retroviruses genomes. 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(1993) Hepatitis B virus: significance of naturally occurring mutants. Intervirology 35, 40–50. Supplementary material The following material is available from: http://www. blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3650/EJB3650sm.htm Figure S1. Kinetic assay for site-specific nucleotide mis- insertion with 5385T primer. The 5¢-[ 32 P] labeled primer 5385T was annealed to M13mp18 template strand to pro- duce the 3¢-terminal mispairs in the presence of increasing concentrations of single dNTP as indicated, with FPolE or HIV-l RT. Figure S2. Kinetic assay for site-specific nucleotide mis- insertion with 2226A primer. The 5¢-[ 32 P] labeled primer 2226A was annealed to M13mp18 template strand to produce the 3¢-terminal mispairs in the presence of increa- sing concentrations of single dNTP as indicated, with FPolE or HIV-l RT. Figure S3. Kinetic assay for site-specific nucleotide mis- insertion with 1510G primer. The 5¢-[ 32 P] labeled primer 1510G was annealed to M13mp18 template strand to produce the 3¢-terminal mispairs in the presence of increa- sing concentrations of single dNTP as indicated, with FPolE or HIV-l RT. Figure S4. Kinetic assay for site-specific nucleotide mis- insertion with 1212C primer. The 5¢-[ 32 P] labeled primer 1212C was annealed to M13mp18 template strand to pro- duce the 3¢-terminal mispairs in the presence of increasing concentrations of single dNTP as indicated, with FPolE or HIV-l RT. 2936 S. G. Park et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . retroviruses. Keywords: HBV polymerase; HBV; fidelity; misinsertion; mispair; exonuclease. The hepatitis B virus (HBV) is a member of the hepadnavi- ridae, a family of enveloped hepatotropic DNA viruses. The virus. infection remains to be elucidated, but the genetic variations of HBV are possibly related to the infidelity of the HBV polymerase and reverse transcription strategy of HBV. Fidelity of DNA synthesis is a major. concentration of dNTP, the observed rate of deoxynucleotide incorporation (V obs )wasdeterminedby dividing the relative amount of the extended product by the incubation time. The observed rate of deoxynucleotide incorporation