Journal of Medical Virology 87:601–608 (2015)
Influence of the Basal Core Promoter and Precore Mutation on Replication of Hepatitis B Virus and Antiviral Susceptibility of Different Genotypes Xiu-Ji Cui, Yoo-Kyung Cho, and Byung-Cheol Song* Department of Internal Medicine, School of Medicine, Jeju National University, Korea
Mutations in the basal core promoter (BCP) and precore (PC) regions of the hepatitis B virus (HBV) are more common in genotypes B and C than in genotype A, suggesting that these mutations might affect replication competency depending on genotype. The purpose of the study was to investigate the influence of these mutations on the capacity of HBV for replication and antiviral drug susceptibility according to genotype. Genotypes A, B, and C of HBV strains with a BCP mutation, PC mutation, or BCP þ PC mutation were made by site-directed mutagenesis. Replication competency of each construct and susceptibility to nucleos(t) ide analogues were tested in an Huh7 cell line. In genotype A, the BCP and BCP þ PC mutations increased the viral replication around 6.5 times compared with the wild type, and the PC mutation alone similarly increased the viral replication around three times. In genotypes B and C, all three mutant types increased viral replication to a similar extent, regardless of mutation pattern. Interestingly, the BCP mutation appeared to have a greater effect on viral replication in genotype A than in genotypes B and C. This finding was unexpected because the BCP mutation is more common in HBV genotypes B and C. Moreover, the BCP, PC, and BCP þ PC mutations decreased the sensitivity of HBV to antiviral agents to various degrees (2- to 10-fold) regardless of genotype. In conclusion, BCP and PC mutations increased viral replication regardless of HBV genotype and decreased in vitro antiviral susceptibility to the nucleos(t) ide analogues. J. Med. Virol. 87:601–608, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
chronic hepatitis B; HBV genotypes; basal core promoter mutation; precore mutation; replication competency; nucleos(t)ide analogues
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Hepatitis B virus (HBV), a major cause of liver disease, has infected approximately 2 billion people worldwide, and more than 350 million are chronically infected [Lavanchy, 2004]. During the HBV replication cycle, lack of a proofreading function in the HBV polymerase results in frequent genetic changes in the HBV genome [Hunt et al., 2000]. Among the naturally occurring genetic changes, adenine to thymine mutation at nucleotide 1,762 and/or guanine to adenine mutation at nucleotide 1,764 (A1762T/G1764A) in the basal core promoter (BCP mutation), which controls the transcription of precore mRNA and pregenomic RNA during HBV replication [Yuh et al., 1992; Chen et al., 1995], and guanine to adenine mutation at nucleotide 1,896 (G1896A) in the precore (PC mutation) region are the most common naturally occurring HBV mutations [Hunt et al., 2000]. In vitro, the BCP mutation enhances viral replication and suppresses HBeAg synthesis [Buckwold et al., 1996; Moriyama et al., 1996]. Moreover, the PC mutation, which prevents the production of HBeAg, also increases viral replication in vitro [Chen et al., 2003a; Tacke et al., 2004]. Accordingly, these mutations are associated with HBeAg-negative chronic hepatitis B [Hunt et al., 2000]. Some studies reported that the BCP mutation was associated with the progression of liver disease [Shindo et al., 1999; Song et al., 2006; Yin et al., 2011; Chu et al., 2012; Tseng et al., 2014] and the Abbreviations: BCP, basal core promoter; HBV, hepatitis B virus; NAs, nucleos(t)ide analogues; PC, precore Grant sponsor: National Research Foundation of Korea (NRF); Grant sponsor: Ministry of Education, Science, and Technology; Grant number: KRF-2008-521-E00034. Correspondence to: Byung-Cheol Song, MD, PhD, Department of Internal Medicine, Jeju National University, School of Medicine, Jeju, Korea, Aran 13 Gil 15, 690-716, Republic of Korea. E-mail: [email protected] Accepted 21 October 2014 DOI 10.1002/jmv.24117 Published online 21 January 2015 in Wiley Online Library (wileyonlinelibrary.com).
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Therefore, the present study evaluated if the BCP and/or PC mutations might affect antiviral drug efficacy according to HBV genotype. The purpose of the present study was to evaluate the influence of BCP and/or PC mutations on the capacity of HBV for replication and the susceptibility to nuldeos(t)ide analogues(NAs) according to genotype.
development of hepatocellular carcinoma [Baptista et al., 1999; Kao et al., 2003; Yang et al., 2008; Yuen et al., 2008]. It has been also reported that the BCP and/or PC mutations were associated with severe or fulminant hepatitis [Carman et al., 1991; Omata et al., 1991; Sato et al., 1995; Baumert et al., 1996; Aritomi et al., 1998; Chen et al., 2003b; Xu et al., 2011]. However, other studies refuted this, and these mutations can even be found in asymptomatic HBV carriers [Akarca et al., 1994; Laskus et al., 1995; Chu et al., 1996; Sterneck et al., 1996; Liu et al., 2004]. The frequency of the BCP and/or PC mutations is different according to HBV genotype and subgenotype. These mutations occur frequently in HBV genotype B or C, but are rare in genotype A [Chan et al., 1999; Orito et al., 2001; Chu et al., 2003a]. The BCP and/or PC mutations are associated with higher serum levels of HBV DNA in HBeAg-negative, but not in HBeAgpositive patients with chronic hepatitis B [Chu et al., 2003b]. The high level of HBV DNA after HBeAg-toanti-HBe seroconversion is related to the coexistence of BCP/PC mutations [Peng et al., 2005]. Clinical studies have demonstrated that high serum levels of HBV DNA, an indicator of high viral replication, increases the risk of liver cirrhosis and the development of hepatocellular carcinoma [Yu et al., 2005; Chen et al., 2006; Iloeje et al., 2006]. In addition, serum level of HBV DNA is one of the main predictors of antiviral drug efficacy [Marcellin et al., 2003, 2008; Liaw et al., 2009].
MATERIALS AND METHODS Construction of Plasmid DNA Containing HBV DNA Full-length HBV DNA was isolated from the serum of patients infected with HBV genotypes A, B, or C and amplified by polymerase chain reaction (PCR) using primer pairs (P1/P2) that have SapI restriction site, as reported previously (Table I) [Gunther et al., 1995]. The PCR product was then digested with the SapI (New England Biolabs, Beverly, MA) at 37˚C for at least 16 hr and recovered from 1% agarose gel. The digested PCR products was then ligated into a SapI-digested pHY106 plasmid (a kind gift from Dr. W. Delaney, Gilead, Foster City, CA), a vector that allows the insertion and expression of full-length HBV genomes [Yang et al., 2004]. The constructed pHY106-HBV plasmid DNA was then transformed into Escherichia coli, and the individual recombinant clone was selected and purified using a Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany). The clones were verified to be replication competent.
TABLE I. Primers Used in the Present Study Oligo Name
Genotypes
Primer sequences
For full-length PCR P1 (nt. 1821–1841)
A, B, C
P2 (nt. 1806–1825)
A, B, C
50 -CCG GAA AGC TTG AGC TCT TCT TTT TCA CCT CTG CCT AAT CA-30 50 -CCG GAA AGC TTG AGC TCT TCA AAA AGT TGC ATG GTG CTG G-30
For mutagenesis BCP (Forward, Gt. A & C, nt. 1746–1781) BCP (Reverse, Gt. A& C, nt. 1746–1781)
A,C A,C
BCP (Forward, Gt. B, nt. 1746–1781)
B
BCP (Reverse, Gt. B, nt. 1746–1781)
B
PC (Forward, G1896A, Gt A & C, nt. 1881–1913)
A,C
PC (Reverse, G1896A, Gt. A & C, nt. 1881–1913)
A,C
PC (Forward, G1896A, Gt. B, nt. 1881–1913)
B
PC (Reverse, G1896A, Gt. B, nt. 1881–1913)
B
PC (Foreward, C1858T, Gt. A & B nt. 1842–1875)
A, B
PC (Reverse, C1858, Gt. A & B, nt. 1842–1875)
A, B
BCP, basal core promoter; PC, precore; Gt, genotype; nt, nucleotide.
J. Med. Virol. DOI 10.1002/jmv
50 -GAG GAG ATT AGG TTA ATG ATC TTT GTA TTA GGA GGC-30 50 -GCC TCC TAA TAC AAA GAT CAT TAA CCTAATCTCCTC-30 50 -GAG GAG ATG AGG TTA ATG ATC TTT GTA TTA GGA GGC-30 50 -GCC TCC TAA TAC AAA GAT CATTAA CCT CAT CTC CTC-30 50 -GCC TTG GGT GGC TTT AGG GCA TGG ACA TTG ACC-30 50 -GGT CAA TGT CCA TGC CCT AAA GCC ACC CAA GGC-3 50 -GCC TTG GGT GGC TTT AGG GCA TGG ACA TTG ACC-30 50 -GGT CAA TGT CCA TGC CCT AAA GCC ACC CAA GGC-30 50 -TCT CTT GTA CAT GTC CTA CTG TTC AAG CCT CCA A-30 5’-TTG GAG GCT TGA ACA GTA GGA CAT GTA CAA GAG A-3’
BCP, PC on HBV Replication and Susceptibility to NAs
PCR-Based Site-Directed Mutagenesis To evaluate the influence of the BCP and PC mutations on viral replication and sensitivity to antiviral agents, the BCP mutation was introduced into wild-type pHY106-HBV of each genotype by sitedirected mutagenesis (QuickChange II Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA) according to the manufacturer’s instructions, using the primer pairs presented in Table I. In brief, a PCR was conducted in a tube containing 50 ml of the following: 0.2 mM of each primer, 0.2 mM of each of the 4 dNTPs, 5 ml of 10 reaction buffer, 2.5 units of Pfu Ultra HiFidelity DNA polymerase (Stratagene), and 50 ng of wild-type pHY106-HBV. PCR was programmed for the first incubation of the samples at 95˚C for 30 sec, followed by 12 cycles at 95˚C for 30 sec, 55˚C for 30 sec, and then at 68˚C for 10 min. Then, the PCR product was treated with DpnI and incubated at 37˚C for 2 hr to remove the parental DNA template. To generate the PC mutation in the inserted HBV genome, full-length HBV genome was amplified with a P1/P2 primer pair using the wild-type or the BCP mutant pHY106-HBV construct as templates using TaKaRa LA Taq polymerase (TaKaRa, Shiga, Japan) and ligated into a pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturer’s instructions. Then, the PC or BCP þ PC mutant was generated from the wild-type or BCP mutant pGEM-T Easy vector-HBV construct by site-directed mutagenesis. Subsequently, the pGEM-T Easy vector-HBV harboring the PC and BCP þ PC mutation were digested with SapI and the HBV genome was recovered from agarose gel. Finally, the DNA was religated into SapI-digested pHY106 vector to generate pHY106-HBV PC and pHY106-HBV BCP þ PC. Because the nucleotide at position 1858 is a cytosine that forms a base pair with guanine at position 1,896 in genotypes A and B, the cytosine at position nucleotide 1,858 was changed to thymine prior to performing a PC mutation. Primer pairs used for the PC mutation for each genotype are presented in Table I. Briefly, PCR was conducted in a tube containing 50 ml of the following: 0.2 mM of each primer, 0.2 mM of each of the 4 dNTPs, 25 ml of 2 PCR buffer, 0.5 unit of LA Taq polymerase (TaKaRa LA Taq polymerase with GC buffer) and 50 ng of wild-type pHY106-HBV DNA. PCR was programmed for the first incubation of the samples at 94˚C for 5 min, followed by 15 cycles at 94˚C for 1 min, at 60˚C for 1 min, and then at 72˚C for 3.5 min, with a 10 min extension step at 72˚C. The PCR products were purified from 1% of agarose gel using a QIAquick Gel Extraction Kit (Qiagen GmbH). Then, the purified PCR product was ligated into a pGEM-T Easy vector (Promega) according to the manufacturer’s instructions. All constructs were sequenced to confirm this modification. Cell Culture and Transfection Huh7 cells were grown in a low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% of
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(vol/vol) fetal bovine serum (Gibco, Grand Island, NY) at 37˚C and 5% CO2. To determine the viral replication competency, 8 105 Huh7 cells were seeded in a 10-cm culture dish. After 24 hr post-seeding, cells were transfected with 2 mg of each plasmid DNA using the lipofectamine (Invitrogen, Carlsbad, CA) and harvested after 72 hr transfection. Transfection efficiency was measured by cotransfection with 0.1 mg of pSEAP (Clontech, Shiga, Japan) expressing secreted alkaline phosphatase and measuring this enzyme activity in the culture medium. To test susceptibility to antiviral drugs, 6 105 Huh7 cells were seeded in a 10-cm culture dish. After 24 hr post-seeding, cells were transfected with 2 mg of each plasmid DNA using the lipofectamine. From the following day, the transfected cells were supplemented with fresh medium containing various concentrations of lamivudine (GlaxoSmithKline, Greenford, Middlesex, UK), adefovir dipivoxil (Gilead), entecavir (Bristol-Myers Squibb, New York, NY) and tenofovir disoproxil fumarate (Gilead) every 24 hr for up to 5 days, and then harvested. The antiviral NA drugs, which are normally administered orally, were dissolved in sterilized and deionized water and then filtered through a 0.20 mm syringe filter (Advantec, Tokyo, Japan). The susceptibility of wild-type and mutant-type HBV to antiviral drugs was evaluated using various concentrations of lamivudine (final concentration: 0, 0.01, 0.1, 1 mM), adefovir (final concentration: 0, 0.001, 0.01, 0.1 mM), entecavir (final concentration: 0, 0.00001, 0.0001, 0.001 mM), and tenofovir (final concentration: 0, 0.01, 0.1, 1 mM), and the 50% inhibitory concentration (IC50) determined to compare the relative sensitivity of the viruses. Isolation of Intracellular HBV DNAa in the Core Particle and Southern Blot Analysis Intracellular HBV DNA in core particles was isolated according to a previously described method [Gunther et al., 1995]. In brief, the cells were washed twice with cold phosphate-buffered saline and lysed with 900 ml of lysis buffer (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% Nonidet P40 (Roche Applied Science, Indianapolis, IN) per a 10-cm culture dish. After mixing thoroughly, the lysate was transferred to a 1.5-ml microcentrifuge tube and incubated on ice for 30 min and it was centrifuged at 18,000g for 10 min at 4˚C. Subsequently, the supernatants were transferred to a new microcentrifuge tube and were adjusted to a final concentration of 10 mM MgCl2 and 2 mM CaCl2 and treated with 20 units of DNase I and 100 mg/ml of RNase A (TaKaRa), then incubated at 37˚C for 1 hr. The enzyme was stopped by the addition of EDTA to a final concentration of 25 mM. The HBV capsid was digested with a final concentration of 0.5 mg/ml proteinase K (Amresco, Cleveland, OH) in 1% of SDS for 4 hr at 55˚C. Then, the HBV nucleic acid was purified using a phenol– chloroform extraction method with ethanol precipitation. J. Med. Virol. DOI 10.1002/jmv
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The amount of intracellular HBV replicative intermediates, including immature DNA forms, was determined by 1% agarose gel electrophoresis at low voltage (50 V) and blotting onto a Nylon membrane (Whatman, Over, Cambridge, UK). The blotted DNA was hybridized with a biotin-labeled random primer generated from a full-length HBV DNA using a random prime labeling kit (Pierce, Rockford, IL), and then visualized by exposure to X-ray film. The amount of intracellular HBV intermediates was determined by Southern blotting using a North2South Chemiluminescent Hybridization and Detection kit (Pierce) according to the manufacturer’s instructions. The efficiency of transfection was normalized by cotransfecting with 0.1 mg pSEAP, a plasmid expressing a secreted form of alkaline phosphatase (Great EscAPe SEAP Chemiluminescence Kit version 2.0, Clontech). The density of signals was measured using
Image J software (http://rsbweb.nih.gov/ij/). IC50s were calculated by nonlinear regression using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). RESULTS Influence of BCP and PC Mutations on Intracellular Viral Replication BCP, PC, and BCP þ PC mutations all increased the intracellular viral replication regardless of HBV genotype (Fig. 1). In genotype A, BCP and BCP þ PC mutations increased the viral replication by 6.5- and 7.0-fold compared with the wild type, respectively. PC mutation alone increased the viral replication by 2.8fold compared with the wild type. In genotypes B and C, all three mutant types increased intracellular viral replication to a similar extent, regardless of mutation
Fig. 1. Effect of the BCP, PC, and BCP þ PC mutations on intracellular viral replication. A: Wild- or mutant-type pHY106-HBV were transfected into 8 105 Huh7 cells and the intracellular HBV DNA was determined by Southern blotting. B: The relative intracellular viral replication was determined as fold changes compared with the wild-type strain. WT, wild type; BCP, basal core promoter; PC, precore.
J. Med. Virol. DOI 10.1002/jmv
BCP, PC on HBV Replication and Susceptibility to NAs
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pattern (Genotype B: BCP mutation, 1.7-fold; BCP þ PC mutation, 1.5-fold, PC mutation, 1.6-fold vs. wild type; Genotype C: BCP mutation, 2.5-fold; BCP þ PC mutation, 2.7-fold, PC mutation, 2.6-fold vs. wild type). Susceptibility of Wild-Type and Mutant-Type HBV to NAs Susceptibility of the wild-type HBV to NAs was similar regardless of genotype. IC50 levels for lamivudine were 0.011, 0.013, and 0.012 mM in wild-type HBV genotypes A, B, and C, respectively (Table II and Fig. 2). However, BCP mutation, PC mutation, and BCP þ PC mutation in genotypes A, B, or C all decreased the susceptibility to NAs. IC50 ranged from 2.0-fold to 10.7-fold of the wild-type HBV for the BCP, PC, and BCP þ PC mutations. There were conspicuous differences in the susceptibility to entecavir and tenofovir, which are recommended as the first-line antiviral agents to treat HBV [Lok and McMahon, 2007; European Association for the Study of the Liver, 2012]. In genotype A with a BCP mutation, the IC50s for entecavir and tenofovir were 3.8- and 8.9-fold of the wild type. By contrast, for the PC mutation, IC50s for entecavir and tenofovir were 8.2- and 5.3-fold of the wild type. In genotype B with a BCP mutation, IC50s for entecavir and tenofovir were 3.5- and 2.2-fold of the wild type. By contrast, in PC mutation, IC50s for entecavir and tenofovir were 5.0- and 10.2-fold of the wild type. In genotype C with a PC mutation, IC50s for entecavir and tenofovir were 10.7- and 1.5-fold of the wild type. In genotype C with a BCP mutation, IC50s for entecavir and tenofovir were 7.6- and 3.7-fold of the wild type. In genotype C with a PC mutation, IC50s for entecavir and tenofovir were 10.7- and 1.5-fold.
DISCUSSION This study demonstrated that the BCP mutation increased viral replication regardless of HBV genotype, and this finding is consistent with previous in vitro studies [Buckwold et al., 1996; Moriyama et al., 1996]. The effect of the BCP mutation on viral replication was the most conspicuous for genotype A. A PC mutation increased viral replication in all three genotypes. By contrast with the BCP mutation, the PC mutation had similar effects on the viral replication for each genotype. Generally, the BCP mutation is found more frequently in genotypes B and C than in genotype A. Thus, this study was performed on the hypothesis that the BCP mutation might facilitate viral replication more effectively in HBV genotypes B and C. However, the BCP mutation increased viral replication more effectively in genotype A than in genotype B or C in this study. The exact mechanism of the different effects of the BCP mutation on the on viral replication of the various HBV genotypes remains clear. Previous studies showed some conflicting findings for correlations between the BCP and/or PC mutation and fulminant hepatitis [Carman et al., 1991; Omata et al., 1991; Akarca et al., 1994; Laskus et al., 1995; Sato et al., 1995; Baumert et al., 1996; Chu et al., 1996; Sterneck et al., 1996; Aritomi et al., 1998; Chen et al., 2003b; Liu et al., 2004]. In this study, the BCP mutation with or without the PC mutation increased viral replication dramatically, especially for HBV genotype A. Therefore, an abrupt increase in viral loads associated with the BCP mutation might induce subsequent acute exacerbations of chronic hepatitis B or fulminant hepatitis in certain situations [Jeng et al., 2010; Ohkawa et al., 2010].
TABLE II. Antiviral Susceptibility of Wild Type- and Mutant Type-HBV to Nucleos(t)ide Analogues Lamivudine
Genotype A WT BCP PC BCP þ PC Genotype B WT BCP PC BCP þ PC Genotype C WT BCP PC BCP þ PC
Adefovir
IC50
Fold change
0.011 0.025 0.032 0.071
0.001 0.013 0.006 0.005
1.0 2.4 3.0 6.6
0.013 0.037 0.058 0.045
0.001 0.026 0.016 0.026
0.012 0.028 0.048 0.032
0.001 0.007 0.01 0.01
Entecarvir
Tenofovir
IC50
Fold change
0.0019 0.0068 0.0092 0.0078
0.001 0.002 0.003 0.003
1 3.5 4.5 4.0
1.05E-5 3.98E-5 8.58E-5 5.13E-5
1.7120E-6 3.8723E-5 8.7611E-5 4.7412E-5
1.0 3.8 8.2 4.9
1 2.8 4.5 3.5
0.0027 0.0112 0.0141 0.0136
0.002 0.002 0.005 0.006
1 4.1 5.1 5.0
1.94E-5 6.40E-5 9.16E-5 6.25E-5
7.38E-6 2.46E-6 6.44E-5 1.01E-5
1.0 2.3 3.9 2.6
0.0023 0.0092 0.0105 0.0049
0.001 0.002 0.005 0.002
1 4.6 4 2.0
1.17E-5 8.91E-5 1.26E-4 5.09E-5
1.07E-6 8.12E-5 4.87E-6 2.18E-5
IC50
Fold change
IC50
Fold change
0.021 0.187 0.111 0.045
0.001 0.007 0.002 0.018
1 8.9 5.3 2.1
1 3.5 5.0 3.4
0.019 0.042 0.193 0.113
0.002 0.003 0.08 0.001
1 2.2 10.2 5.9
1.0 7.6 10.7 4.3
0.022 0.082 0.034 0.157
0.013 0.017 0.017 0.012
1 3.7 1.5 7.1
Results are from duplicating experiments. The resistant fold is the IC50 ratio of mutant/WT and present as mean SD. WT, wild-type; BCP, basal core promoter; PC, precore.
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Fig. 2. Sensitivity of wild-type and mutant-type HBV A, B, or C genotypes to antiviral NA drugs. Replicating capacity of wild-type and mutant-type HBV in various concentrations of antiviral drugs was determined by Southern blotting and the density of signal were normalized to that of the untreated strain of either wild-type or mutant-type HBV. The data were used to determine IC50s by a nonlinear regression method. WT, wild type; BCP, basal core promoter; PC, precore.
J. Med. Virol. DOI 10.1002/jmv
BCP, PC on HBV Replication and Susceptibility to NAs
The present study also evaluated the effect of BCP and PC mutation on the sensitivity of the various HBV genotypes to NAs. IC50s for lamivudine, adefovir, entecavir, and tenofovir were higher in BCP and/ or PC-mutant HBV strains compared with wild-type HBV. This finding suggests that a higher concentration or more potent antiviral agent might be needed to suppress the viral replication of these mutant strains effectively. Several studies reported different response to antiviral drugs according to HBV genotype. Although HBV genotype C has a poor response to interferonbased therapy compared with genotypes A or B [Kao et al., 2000; Wai et al., 2002; Flink et al., 2006], HBV genotypes have little different effect on treatment with NAs [Yuen et al., 2003; Wiegand et al., 2008]. This study showed conspicuously different susceptibility to entecavir and tenofovir. For example, in genotype C with a PC mutant, the IC50s for entecavir and tenofovir were 10.7- and 1.5-fold those for the wild type. By contrast, in genotype B with a PC mutant, the IC50s for entecavir and tenofovir were 5and 10.2-fold of those for the wild type. This finding suggests that HBV genotype C with a PC mutant is more susceptible to tenofovir than entecavir. However, HBV genotype B with a PC mutant was found to be more susceptible to entecavir. Therefore, clinical studies may clarify this issue. In conclusion, BCP and PC mutations increased the viral replication competence regardless of HBV genotype in vitro. The HBV with a BCP mutation affects different replication competency depending on HBV genotype. However, HBVs with a PC mutation show similar replication competence regardless of genotype. REFERENCES Akarca US, Greene S, Lok AS. 1994. Detection of precore hepatitis B virus mutants in asymptomatic HBsAg-positive family members. Hepatology 19:1366–1370. Aritomi T, Yatsuhashi H, Fujino T, Yamasaki K, Inoue O, Koga M, Kato Y, Yano M. 1998. Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan. J Gastroenterol Hepatol 13:1125–1132. Baptista M, Kramvis A, Kew MC. 1999. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 29:946–953. Baumert TF, Rogers SA, Hasegawa K, Liang TJ. 1996. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J Clin Invest 98:2268–2276. Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. 1996. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 70:5845–5851. Carman WF, Fagan EA, Hadziyannis S, Karayiannis P, Tassopoulos NC, Williams R, Thomas HC. 1991. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 14:219–222. Chan HL, Hussain M, Lok AS. 1999. Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 29:976–984.
607 Chen IH, Huang CJ, Ting LP. 1995. Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus. J Virol 69:3647–3657. Chen RY, Edwards R, Shaw T, Colledge D, Delaney WE, Isom H, Bowden S, Desmond P, Locarnini SA. 2003a. Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine-resistant HBV in vitro. Hepatology 37:27–35. Chen Y, Michitaka K, Matsubara H, Yamamoto K, Horiike N, Onji M. 2003b. Complete genome sequence of hepatitis B virus (HBV) from a patient with fulminant hepatitis without precore and core promoter mutations: Comparison with HBV from a patient with acute hepatitis infected from the same infectious source. J Hepatol 38:84–90. Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN, Huang GT, Iloeje UH. 2006. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA 295:65–73. Chu CJ, Keeffe EB, Han SH, Perrillo RP, Min AD, Soldevila-Pico C, Carey W, Brown RS Jr., Luketic VA, Terrault N, Lok AS. 2003a. Hepatitis B virus genotypes in the United States: Results of a nationwide study. Gastroenterology 125:444–451. Chu CJ, Keeffe EB, Han SH, Perrillo RP, Min AD, Soldevila-Pico C, Carey W, Brown RS Jr., Luketic VA, Terrault N, Lok AS. 2003b. Prevalence of HBV precore/core promoter variants in the United States. Hepatology 38:619–628. Chu CM, Lin CC, Chen YC, Jeng WJ, Lin SM, Liaw YF. 2012. Basal core promoter mutation is associated with progression to cirrhosis rather than hepatocellular carcinoma in chronic hepatitis B virus infection. Br J Cancer 107:2010–2015. Chu CM, Yeh CT, Chiu CT, Sheen IS, Liaw YF. 1996. Precore mutant of hepatitis B virus prevails in acute and chronic infections in an area in which hepatitis B is endemic. J Clin Microbiol 34:1815–1818. European Association for the Study of the Liver. 2012. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J Hepatol 57:167–185. Flink HJ, van Zonneveld M, Hansen BE, de Man RA, Schalm SW, Janssen HL. 2006. Treatment with Peg-interferon alpha-2b for HBeAg-positive chronic hepatitis B: HBsAg loss is associated with HBV genotype. Am J Gastroenterol 101:297–303. Gunther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. 1995. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69:5437–5444. Hunt CM, McGill JM, Allen MI, Condreay LD. 2000. Clinical relevance of hepatitis B viral mutations. Hepatology 31:1037–1044. Iloeje UH, Yang HI, Su J, Jen CL, You SL, Chen CJ. 2006. Risk evaluation of viral load E, associated liver, disease/cancer-in HBVSG: Predicting cirrhosis risk based on the level of circulating hepatitis b viral load. Gastroenterology 130:678–686. Jeng WJ, Sheen IS, Liaw YF. 2010. Hepatitis B virus DNA level predicts hepatic decompensation in patients with acute exacerbation of chronic hepatitis B. Clin Gastroenterol Hepatol 8:541–545. Kao JH, Chen PJ, Lai MY, Chen DS. 2003. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology 124:327– 334. Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. 2000. Hepatitis B genotypes and the response to interferon therapy. J Hepatol 33:998–1002. Laskus T, Rakela J, Nowicki MJ, Persing DH. 1995. Hepatitis B virus core promoter sequence analysis in fulminant and chronic hepatitis B. Gastroenterology 109:1618–1623. Lavanchy D. 2004. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 11:97–107. Liaw YF, Gane E, Leung N, Zeuzem S, Wang Y, Lai CL, Heathcote EJ, Manns M, Bzowej N, Niu J, Han SH, Hwang SG, Cakaloglu Y, Tong MJ, Papatheodoridis G, Chen Y, Brown NA, Albanis E, Galil K, Naoumov NV. 2009. 2-Year GLOBE trial results: Telbivudine is superior to lamivudine in patients with chronic hepatitis B. Gastroenterology 136:486–495. Liu CJ, Kao JH, Lai MY, Chen PJ, Chen DS. 2004. Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis. J Med Virol 72:545–550. Lok AS, McMahon BJ. 2007. Chronic hepatitis B. Hepatology 45:507–539.
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608 Marcellin P, Chang TT, Lim SG, Tong MJ, Sievert W, Shiffman ML, Jeffers L, Goodman Z, Wulfsohn MS, Xiong S, Fry J, Brosgart CL. 2003. Adefovir dipivoxil for the treatment of hepatitis B e antigenpositive chronic hepatitis B. N Engl J Med 348:808–816. Marcellin P, Heathcote EJ, Buti M, Gane E, de Man RA, Krastev Z, Germanidis G, Lee SS, Flisiak R, Kaita K, Manns M, Kotzev I, Tchernev K, Buggisch P, Weilert F, Kurdas OO, Shiffman ML, Trinh H, Washington MK, Sorbel J, Anderson J, Snow-Lampart A, Mondou E, Quinn J, Rousseau F. 2008. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N Engl J Med 359:2442–2455. Moriyama K, Okamoto H, Tsuda F, Mayumi M. 1996. Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precorecore promoter with sequences associated with e antigen-seronegative persistent infections. Virology 226:269–280. Ohkawa K, Takehara T, Ishida H, Kodama T, Shimizu S, Hikita H, Yamamoto M, Kohga K, Sasakawa A, Uemura A, Sakamori R, Yamaguchi S, Li W, Hosui A, Miyagi T, Tatsumi T, Katayama K, Hayashi N. 2010. Fatal exacerbation of type B chronic hepatitis triggered by changes in relaxed circular viral DNA synthesis and virion secretion. Biochem Biophys Res Commun 394:87–93. Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. 1991. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med 324:1699–1704. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, Okanoue T, Yotsuyanagi H, Iino S. 2001. A casecontrol study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C Japan HBV Genotype Research Group. Hepatology 33:218–223. Peng XM, Huang GM, Li JG, Huang YS, Mei YY, Gao ZL. 2005. High level of hepatitis B virus DNA after HBeAg-to-anti-HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter. World J Gastroenterol 11:3131–3134. Sato S, Suzuki K, Akahane Y, Akamatsu K, Akiyama K, Yunomura K, Tsuda F, Tanaka T, Okamoto H, Miyakawa Y, Mayumi M. 1995. Hepatitis B virus strains with mutations in the core promoter in patients with fulminant hepatitis. Ann Intern Med 122:241–248. Shindo M, Hamada K, Koya S, Sokawa Y, Okuno T. 1999. The clinical significance of core promoter and precore mutations during the natural course and interferon therapy in patients with chronic hepatitis B. Am J Gastroenterol 94:2237–2245. Song BC, Cui XJ, Kim HU, Cho YK. 2006. Sequential accumulation of the basal core promoter and the precore mutations in the progression of hepatitis B virus-related chronic liver disease. Intervirology 49:266–273. Sterneck M, Gunther S, Santantonio T, Fischer L, Broelsch CE, Greten H, Will H. 1996. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. Hepatology 24:300–306.
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Cui et al. Tacke F, Gehrke C, Luedde T, Heim A, Manns MP, Trautwein C. 2004. Basal core promoter and precore mutations in the hepatitis B virus genome enhance replication efficacy of Lamivudine-resistant mutants. J Virol 78:8524–8535. Tseng TC, Liu CJ, Yang HC, Chen CL, Yang WT, Tsai CS, Kuo SF, Verbree FC, Su TH, Wang CC, Liu CH, Chen PJ, Chen DS, Kao JH. 2014. Higher proportion of viral basal core promoter mutant increases the risk of liver cirrhosis in hepatitis B carriers. Gut. DOI: 10.1136/gutjnl-2014-306977 Wai CT, Chu CJ, Hussain M, Lok AS. 2002. HBV genotype B is associated with better response to interferon therapy in HBeAg (þ) chronic hepatitis than genotype C. Hepatology 36:1425– 1430. Wiegand J, Hasenclever D, Tillmann HL. 2008. Should treatment of hepatitis B depend on hepatitis B virus genotypes? A hypothesis generated from an explorative analysis of published evidence. Antivir Ther 13:211–220. Xu Z, Ren X, Liu Y, Li X, Bai S, Zhong Y, Wang L, Mao P, Wang H, Xin S, Wong VW, Chan HL, Zoulim F, Xu D. 2011. Association of hepatitis B virus mutations in basal core promoter and precore regions with severity of liver disease: An investigation of 793 Chinese patients with mild and severe chronic hepatitis B and acute-on-chronic liver failure. J Gastroenterol 46:391–400. Yang H, Westland C, Xiong S, Delaney WEt. 2004. In vitro antiviral susceptibility of full-length clinical hepatitis B virus isolates cloned with a novel expression vector. Antiviral Res 61:27–36. Yang HI, Yeh SH, Chen PJ, Iloeje UH, Jen CL, Su J, Wang LY, Lu SN, You SL, Chen DS, Liaw YF, Chen CJ. 2008. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma. J Natl Cancer Inst 100:11 34–1143. Yin J, Xie J, Liu S, Zhang H, Han L, Lu W, Shen Q, Xu G, Dong H, Shen J, Zhang J, Han J, Wang L, Liu Y, Wang F, Zhao J, Zhang Q, Ni W, Wang H, Cao G. 2011. Association between the various mutations in viral core promoter region to different stages of hepatitis B, ranging of asymptomatic carrier state to hepatocellular carcinoma. Am J Gastroenterol 106:81–92. Yu MW, Yeh SH, Chen PJ, Liaw YF, Lin CL, Liu CJ, Shih WL, Kao JH, Chen DS, Chen CJ. 2005. Hepatitis B virus genotype and DNA level and hepatocellular carcinoma: A prospective study in men. J Natl Cancer Inst 97:265–272. Yuen MF, Tanaka Y, Lai CL. 2003. Hepatitis B genotypes in chronic hepatitis B and lamivudine therapy. Intervirology 46:373–376. Yuen MF, Tanaka Y, Shinkai N, Poon RT, But DY, Fong DY, Fung J, Wong DK, Yuen JC, Mizokami M, Lai CL. 2008. Risk for hepatocellular carcinoma with respect to hepatitis B virus genotypes B/C, specific mutations of enhancer II/core promoter/ precore regions and HBV DNA levels. Gut 57:98–102. Yuh CH, Chang YL, Ting LP. 1992. Transcriptional regulation of precore and pregenomic RNAs of hepatitis B virus. J Virol 66:4073–4084.
Influence of the Basal Core Promoter and Precore Mutation on Replication of Hepatitis B Virus and Antiviral Susceptibility of Different Genotypes Xiu-Ji Cui, Yoo-Kyung Cho, and Byung-Cheol Song* Department of Internal Medicine, School of Medicine, Jeju National University, Korea
Mutations in the basal core promoter (BCP) and precore (PC) regions of the hepatitis B virus (HBV) are more common in genotypes B and C than in genotype A, suggesting that these mutations might affect replication competency depending on genotype. The purpose of the study was to investigate the influence of these mutations on the capacity of HBV for replication and antiviral drug susceptibility according to genotype. Genotypes A, B, and C of HBV strains with a BCP mutation, PC mutation, or BCP þ PC mutation were made by site-directed mutagenesis. Replication competency of each construct and susceptibility to nucleos(t) ide analogues were tested in an Huh7 cell line. In genotype A, the BCP and BCP þ PC mutations increased the viral replication around 6.5 times compared with the wild type, and the PC mutation alone similarly increased the viral replication around three times. In genotypes B and C, all three mutant types increased viral replication to a similar extent, regardless of mutation pattern. Interestingly, the BCP mutation appeared to have a greater effect on viral replication in genotype A than in genotypes B and C. This finding was unexpected because the BCP mutation is more common in HBV genotypes B and C. Moreover, the BCP, PC, and BCP þ PC mutations decreased the sensitivity of HBV to antiviral agents to various degrees (2- to 10-fold) regardless of genotype. In conclusion, BCP and PC mutations increased viral replication regardless of HBV genotype and decreased in vitro antiviral susceptibility to the nucleos(t) ide analogues. J. Med. Virol. 87:601–608, 2015. # 2015 Wiley Periodicals, Inc.
KEY WORDS:
chronic hepatitis B; HBV genotypes; basal core promoter mutation; precore mutation; replication competency; nucleos(t)ide analogues
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Hepatitis B virus (HBV), a major cause of liver disease, has infected approximately 2 billion people worldwide, and more than 350 million are chronically infected [Lavanchy, 2004]. During the HBV replication cycle, lack of a proofreading function in the HBV polymerase results in frequent genetic changes in the HBV genome [Hunt et al., 2000]. Among the naturally occurring genetic changes, adenine to thymine mutation at nucleotide 1,762 and/or guanine to adenine mutation at nucleotide 1,764 (A1762T/G1764A) in the basal core promoter (BCP mutation), which controls the transcription of precore mRNA and pregenomic RNA during HBV replication [Yuh et al., 1992; Chen et al., 1995], and guanine to adenine mutation at nucleotide 1,896 (G1896A) in the precore (PC mutation) region are the most common naturally occurring HBV mutations [Hunt et al., 2000]. In vitro, the BCP mutation enhances viral replication and suppresses HBeAg synthesis [Buckwold et al., 1996; Moriyama et al., 1996]. Moreover, the PC mutation, which prevents the production of HBeAg, also increases viral replication in vitro [Chen et al., 2003a; Tacke et al., 2004]. Accordingly, these mutations are associated with HBeAg-negative chronic hepatitis B [Hunt et al., 2000]. Some studies reported that the BCP mutation was associated with the progression of liver disease [Shindo et al., 1999; Song et al., 2006; Yin et al., 2011; Chu et al., 2012; Tseng et al., 2014] and the Abbreviations: BCP, basal core promoter; HBV, hepatitis B virus; NAs, nucleos(t)ide analogues; PC, precore Grant sponsor: National Research Foundation of Korea (NRF); Grant sponsor: Ministry of Education, Science, and Technology; Grant number: KRF-2008-521-E00034. Correspondence to: Byung-Cheol Song, MD, PhD, Department of Internal Medicine, Jeju National University, School of Medicine, Jeju, Korea, Aran 13 Gil 15, 690-716, Republic of Korea. E-mail: [email protected] Accepted 21 October 2014 DOI 10.1002/jmv.24117 Published online 21 January 2015 in Wiley Online Library (wileyonlinelibrary.com).
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Therefore, the present study evaluated if the BCP and/or PC mutations might affect antiviral drug efficacy according to HBV genotype. The purpose of the present study was to evaluate the influence of BCP and/or PC mutations on the capacity of HBV for replication and the susceptibility to nuldeos(t)ide analogues(NAs) according to genotype.
development of hepatocellular carcinoma [Baptista et al., 1999; Kao et al., 2003; Yang et al., 2008; Yuen et al., 2008]. It has been also reported that the BCP and/or PC mutations were associated with severe or fulminant hepatitis [Carman et al., 1991; Omata et al., 1991; Sato et al., 1995; Baumert et al., 1996; Aritomi et al., 1998; Chen et al., 2003b; Xu et al., 2011]. However, other studies refuted this, and these mutations can even be found in asymptomatic HBV carriers [Akarca et al., 1994; Laskus et al., 1995; Chu et al., 1996; Sterneck et al., 1996; Liu et al., 2004]. The frequency of the BCP and/or PC mutations is different according to HBV genotype and subgenotype. These mutations occur frequently in HBV genotype B or C, but are rare in genotype A [Chan et al., 1999; Orito et al., 2001; Chu et al., 2003a]. The BCP and/or PC mutations are associated with higher serum levels of HBV DNA in HBeAg-negative, but not in HBeAgpositive patients with chronic hepatitis B [Chu et al., 2003b]. The high level of HBV DNA after HBeAg-toanti-HBe seroconversion is related to the coexistence of BCP/PC mutations [Peng et al., 2005]. Clinical studies have demonstrated that high serum levels of HBV DNA, an indicator of high viral replication, increases the risk of liver cirrhosis and the development of hepatocellular carcinoma [Yu et al., 2005; Chen et al., 2006; Iloeje et al., 2006]. In addition, serum level of HBV DNA is one of the main predictors of antiviral drug efficacy [Marcellin et al., 2003, 2008; Liaw et al., 2009].
MATERIALS AND METHODS Construction of Plasmid DNA Containing HBV DNA Full-length HBV DNA was isolated from the serum of patients infected with HBV genotypes A, B, or C and amplified by polymerase chain reaction (PCR) using primer pairs (P1/P2) that have SapI restriction site, as reported previously (Table I) [Gunther et al., 1995]. The PCR product was then digested with the SapI (New England Biolabs, Beverly, MA) at 37˚C for at least 16 hr and recovered from 1% agarose gel. The digested PCR products was then ligated into a SapI-digested pHY106 plasmid (a kind gift from Dr. W. Delaney, Gilead, Foster City, CA), a vector that allows the insertion and expression of full-length HBV genomes [Yang et al., 2004]. The constructed pHY106-HBV plasmid DNA was then transformed into Escherichia coli, and the individual recombinant clone was selected and purified using a Qiagen Plasmid Midi Kit (Qiagen, Hilden, Germany). The clones were verified to be replication competent.
TABLE I. Primers Used in the Present Study Oligo Name
Genotypes
Primer sequences
For full-length PCR P1 (nt. 1821–1841)
A, B, C
P2 (nt. 1806–1825)
A, B, C
50 -CCG GAA AGC TTG AGC TCT TCT TTT TCA CCT CTG CCT AAT CA-30 50 -CCG GAA AGC TTG AGC TCT TCA AAA AGT TGC ATG GTG CTG G-30
For mutagenesis BCP (Forward, Gt. A & C, nt. 1746–1781) BCP (Reverse, Gt. A& C, nt. 1746–1781)
A,C A,C
BCP (Forward, Gt. B, nt. 1746–1781)
B
BCP (Reverse, Gt. B, nt. 1746–1781)
B
PC (Forward, G1896A, Gt A & C, nt. 1881–1913)
A,C
PC (Reverse, G1896A, Gt. A & C, nt. 1881–1913)
A,C
PC (Forward, G1896A, Gt. B, nt. 1881–1913)
B
PC (Reverse, G1896A, Gt. B, nt. 1881–1913)
B
PC (Foreward, C1858T, Gt. A & B nt. 1842–1875)
A, B
PC (Reverse, C1858, Gt. A & B, nt. 1842–1875)
A, B
BCP, basal core promoter; PC, precore; Gt, genotype; nt, nucleotide.
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50 -GAG GAG ATT AGG TTA ATG ATC TTT GTA TTA GGA GGC-30 50 -GCC TCC TAA TAC AAA GAT CAT TAA CCTAATCTCCTC-30 50 -GAG GAG ATG AGG TTA ATG ATC TTT GTA TTA GGA GGC-30 50 -GCC TCC TAA TAC AAA GAT CATTAA CCT CAT CTC CTC-30 50 -GCC TTG GGT GGC TTT AGG GCA TGG ACA TTG ACC-30 50 -GGT CAA TGT CCA TGC CCT AAA GCC ACC CAA GGC-3 50 -GCC TTG GGT GGC TTT AGG GCA TGG ACA TTG ACC-30 50 -GGT CAA TGT CCA TGC CCT AAA GCC ACC CAA GGC-30 50 -TCT CTT GTA CAT GTC CTA CTG TTC AAG CCT CCA A-30 5’-TTG GAG GCT TGA ACA GTA GGA CAT GTA CAA GAG A-3’
BCP, PC on HBV Replication and Susceptibility to NAs
PCR-Based Site-Directed Mutagenesis To evaluate the influence of the BCP and PC mutations on viral replication and sensitivity to antiviral agents, the BCP mutation was introduced into wild-type pHY106-HBV of each genotype by sitedirected mutagenesis (QuickChange II Site-Directed Mutagenesis Kit; Stratagene, La Jolla, CA) according to the manufacturer’s instructions, using the primer pairs presented in Table I. In brief, a PCR was conducted in a tube containing 50 ml of the following: 0.2 mM of each primer, 0.2 mM of each of the 4 dNTPs, 5 ml of 10 reaction buffer, 2.5 units of Pfu Ultra HiFidelity DNA polymerase (Stratagene), and 50 ng of wild-type pHY106-HBV. PCR was programmed for the first incubation of the samples at 95˚C for 30 sec, followed by 12 cycles at 95˚C for 30 sec, 55˚C for 30 sec, and then at 68˚C for 10 min. Then, the PCR product was treated with DpnI and incubated at 37˚C for 2 hr to remove the parental DNA template. To generate the PC mutation in the inserted HBV genome, full-length HBV genome was amplified with a P1/P2 primer pair using the wild-type or the BCP mutant pHY106-HBV construct as templates using TaKaRa LA Taq polymerase (TaKaRa, Shiga, Japan) and ligated into a pGEM-T Easy vector (Promega, Madison, WI) according to the manufacturer’s instructions. Then, the PC or BCP þ PC mutant was generated from the wild-type or BCP mutant pGEM-T Easy vector-HBV construct by site-directed mutagenesis. Subsequently, the pGEM-T Easy vector-HBV harboring the PC and BCP þ PC mutation were digested with SapI and the HBV genome was recovered from agarose gel. Finally, the DNA was religated into SapI-digested pHY106 vector to generate pHY106-HBV PC and pHY106-HBV BCP þ PC. Because the nucleotide at position 1858 is a cytosine that forms a base pair with guanine at position 1,896 in genotypes A and B, the cytosine at position nucleotide 1,858 was changed to thymine prior to performing a PC mutation. Primer pairs used for the PC mutation for each genotype are presented in Table I. Briefly, PCR was conducted in a tube containing 50 ml of the following: 0.2 mM of each primer, 0.2 mM of each of the 4 dNTPs, 25 ml of 2 PCR buffer, 0.5 unit of LA Taq polymerase (TaKaRa LA Taq polymerase with GC buffer) and 50 ng of wild-type pHY106-HBV DNA. PCR was programmed for the first incubation of the samples at 94˚C for 5 min, followed by 15 cycles at 94˚C for 1 min, at 60˚C for 1 min, and then at 72˚C for 3.5 min, with a 10 min extension step at 72˚C. The PCR products were purified from 1% of agarose gel using a QIAquick Gel Extraction Kit (Qiagen GmbH). Then, the purified PCR product was ligated into a pGEM-T Easy vector (Promega) according to the manufacturer’s instructions. All constructs were sequenced to confirm this modification. Cell Culture and Transfection Huh7 cells were grown in a low-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% of
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(vol/vol) fetal bovine serum (Gibco, Grand Island, NY) at 37˚C and 5% CO2. To determine the viral replication competency, 8 105 Huh7 cells were seeded in a 10-cm culture dish. After 24 hr post-seeding, cells were transfected with 2 mg of each plasmid DNA using the lipofectamine (Invitrogen, Carlsbad, CA) and harvested after 72 hr transfection. Transfection efficiency was measured by cotransfection with 0.1 mg of pSEAP (Clontech, Shiga, Japan) expressing secreted alkaline phosphatase and measuring this enzyme activity in the culture medium. To test susceptibility to antiviral drugs, 6 105 Huh7 cells were seeded in a 10-cm culture dish. After 24 hr post-seeding, cells were transfected with 2 mg of each plasmid DNA using the lipofectamine. From the following day, the transfected cells were supplemented with fresh medium containing various concentrations of lamivudine (GlaxoSmithKline, Greenford, Middlesex, UK), adefovir dipivoxil (Gilead), entecavir (Bristol-Myers Squibb, New York, NY) and tenofovir disoproxil fumarate (Gilead) every 24 hr for up to 5 days, and then harvested. The antiviral NA drugs, which are normally administered orally, were dissolved in sterilized and deionized water and then filtered through a 0.20 mm syringe filter (Advantec, Tokyo, Japan). The susceptibility of wild-type and mutant-type HBV to antiviral drugs was evaluated using various concentrations of lamivudine (final concentration: 0, 0.01, 0.1, 1 mM), adefovir (final concentration: 0, 0.001, 0.01, 0.1 mM), entecavir (final concentration: 0, 0.00001, 0.0001, 0.001 mM), and tenofovir (final concentration: 0, 0.01, 0.1, 1 mM), and the 50% inhibitory concentration (IC50) determined to compare the relative sensitivity of the viruses. Isolation of Intracellular HBV DNAa in the Core Particle and Southern Blot Analysis Intracellular HBV DNA in core particles was isolated according to a previously described method [Gunther et al., 1995]. In brief, the cells were washed twice with cold phosphate-buffered saline and lysed with 900 ml of lysis buffer (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% Nonidet P40 (Roche Applied Science, Indianapolis, IN) per a 10-cm culture dish. After mixing thoroughly, the lysate was transferred to a 1.5-ml microcentrifuge tube and incubated on ice for 30 min and it was centrifuged at 18,000g for 10 min at 4˚C. Subsequently, the supernatants were transferred to a new microcentrifuge tube and were adjusted to a final concentration of 10 mM MgCl2 and 2 mM CaCl2 and treated with 20 units of DNase I and 100 mg/ml of RNase A (TaKaRa), then incubated at 37˚C for 1 hr. The enzyme was stopped by the addition of EDTA to a final concentration of 25 mM. The HBV capsid was digested with a final concentration of 0.5 mg/ml proteinase K (Amresco, Cleveland, OH) in 1% of SDS for 4 hr at 55˚C. Then, the HBV nucleic acid was purified using a phenol– chloroform extraction method with ethanol precipitation. J. Med. Virol. DOI 10.1002/jmv
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The amount of intracellular HBV replicative intermediates, including immature DNA forms, was determined by 1% agarose gel electrophoresis at low voltage (50 V) and blotting onto a Nylon membrane (Whatman, Over, Cambridge, UK). The blotted DNA was hybridized with a biotin-labeled random primer generated from a full-length HBV DNA using a random prime labeling kit (Pierce, Rockford, IL), and then visualized by exposure to X-ray film. The amount of intracellular HBV intermediates was determined by Southern blotting using a North2South Chemiluminescent Hybridization and Detection kit (Pierce) according to the manufacturer’s instructions. The efficiency of transfection was normalized by cotransfecting with 0.1 mg pSEAP, a plasmid expressing a secreted form of alkaline phosphatase (Great EscAPe SEAP Chemiluminescence Kit version 2.0, Clontech). The density of signals was measured using
Image J software (http://rsbweb.nih.gov/ij/). IC50s were calculated by nonlinear regression using GraphPad Prism 6 software (GraphPad Software, San Diego, CA). RESULTS Influence of BCP and PC Mutations on Intracellular Viral Replication BCP, PC, and BCP þ PC mutations all increased the intracellular viral replication regardless of HBV genotype (Fig. 1). In genotype A, BCP and BCP þ PC mutations increased the viral replication by 6.5- and 7.0-fold compared with the wild type, respectively. PC mutation alone increased the viral replication by 2.8fold compared with the wild type. In genotypes B and C, all three mutant types increased intracellular viral replication to a similar extent, regardless of mutation
Fig. 1. Effect of the BCP, PC, and BCP þ PC mutations on intracellular viral replication. A: Wild- or mutant-type pHY106-HBV were transfected into 8 105 Huh7 cells and the intracellular HBV DNA was determined by Southern blotting. B: The relative intracellular viral replication was determined as fold changes compared with the wild-type strain. WT, wild type; BCP, basal core promoter; PC, precore.
J. Med. Virol. DOI 10.1002/jmv
BCP, PC on HBV Replication and Susceptibility to NAs
605
pattern (Genotype B: BCP mutation, 1.7-fold; BCP þ PC mutation, 1.5-fold, PC mutation, 1.6-fold vs. wild type; Genotype C: BCP mutation, 2.5-fold; BCP þ PC mutation, 2.7-fold, PC mutation, 2.6-fold vs. wild type). Susceptibility of Wild-Type and Mutant-Type HBV to NAs Susceptibility of the wild-type HBV to NAs was similar regardless of genotype. IC50 levels for lamivudine were 0.011, 0.013, and 0.012 mM in wild-type HBV genotypes A, B, and C, respectively (Table II and Fig. 2). However, BCP mutation, PC mutation, and BCP þ PC mutation in genotypes A, B, or C all decreased the susceptibility to NAs. IC50 ranged from 2.0-fold to 10.7-fold of the wild-type HBV for the BCP, PC, and BCP þ PC mutations. There were conspicuous differences in the susceptibility to entecavir and tenofovir, which are recommended as the first-line antiviral agents to treat HBV [Lok and McMahon, 2007; European Association for the Study of the Liver, 2012]. In genotype A with a BCP mutation, the IC50s for entecavir and tenofovir were 3.8- and 8.9-fold of the wild type. By contrast, for the PC mutation, IC50s for entecavir and tenofovir were 8.2- and 5.3-fold of the wild type. In genotype B with a BCP mutation, IC50s for entecavir and tenofovir were 3.5- and 2.2-fold of the wild type. By contrast, in PC mutation, IC50s for entecavir and tenofovir were 5.0- and 10.2-fold of the wild type. In genotype C with a PC mutation, IC50s for entecavir and tenofovir were 10.7- and 1.5-fold of the wild type. In genotype C with a BCP mutation, IC50s for entecavir and tenofovir were 7.6- and 3.7-fold of the wild type. In genotype C with a PC mutation, IC50s for entecavir and tenofovir were 10.7- and 1.5-fold.
DISCUSSION This study demonstrated that the BCP mutation increased viral replication regardless of HBV genotype, and this finding is consistent with previous in vitro studies [Buckwold et al., 1996; Moriyama et al., 1996]. The effect of the BCP mutation on viral replication was the most conspicuous for genotype A. A PC mutation increased viral replication in all three genotypes. By contrast with the BCP mutation, the PC mutation had similar effects on the viral replication for each genotype. Generally, the BCP mutation is found more frequently in genotypes B and C than in genotype A. Thus, this study was performed on the hypothesis that the BCP mutation might facilitate viral replication more effectively in HBV genotypes B and C. However, the BCP mutation increased viral replication more effectively in genotype A than in genotype B or C in this study. The exact mechanism of the different effects of the BCP mutation on the on viral replication of the various HBV genotypes remains clear. Previous studies showed some conflicting findings for correlations between the BCP and/or PC mutation and fulminant hepatitis [Carman et al., 1991; Omata et al., 1991; Akarca et al., 1994; Laskus et al., 1995; Sato et al., 1995; Baumert et al., 1996; Chu et al., 1996; Sterneck et al., 1996; Aritomi et al., 1998; Chen et al., 2003b; Liu et al., 2004]. In this study, the BCP mutation with or without the PC mutation increased viral replication dramatically, especially for HBV genotype A. Therefore, an abrupt increase in viral loads associated with the BCP mutation might induce subsequent acute exacerbations of chronic hepatitis B or fulminant hepatitis in certain situations [Jeng et al., 2010; Ohkawa et al., 2010].
TABLE II. Antiviral Susceptibility of Wild Type- and Mutant Type-HBV to Nucleos(t)ide Analogues Lamivudine
Genotype A WT BCP PC BCP þ PC Genotype B WT BCP PC BCP þ PC Genotype C WT BCP PC BCP þ PC
Adefovir
IC50
Fold change
0.011 0.025 0.032 0.071
0.001 0.013 0.006 0.005
1.0 2.4 3.0 6.6
0.013 0.037 0.058 0.045
0.001 0.026 0.016 0.026
0.012 0.028 0.048 0.032
0.001 0.007 0.01 0.01
Entecarvir
Tenofovir
IC50
Fold change
0.0019 0.0068 0.0092 0.0078
0.001 0.002 0.003 0.003
1 3.5 4.5 4.0
1.05E-5 3.98E-5 8.58E-5 5.13E-5
1.7120E-6 3.8723E-5 8.7611E-5 4.7412E-5
1.0 3.8 8.2 4.9
1 2.8 4.5 3.5
0.0027 0.0112 0.0141 0.0136
0.002 0.002 0.005 0.006
1 4.1 5.1 5.0
1.94E-5 6.40E-5 9.16E-5 6.25E-5
7.38E-6 2.46E-6 6.44E-5 1.01E-5
1.0 2.3 3.9 2.6
0.0023 0.0092 0.0105 0.0049
0.001 0.002 0.005 0.002
1 4.6 4 2.0
1.17E-5 8.91E-5 1.26E-4 5.09E-5
1.07E-6 8.12E-5 4.87E-6 2.18E-5
IC50
Fold change
IC50
Fold change
0.021 0.187 0.111 0.045
0.001 0.007 0.002 0.018
1 8.9 5.3 2.1
1 3.5 5.0 3.4
0.019 0.042 0.193 0.113
0.002 0.003 0.08 0.001
1 2.2 10.2 5.9
1.0 7.6 10.7 4.3
0.022 0.082 0.034 0.157
0.013 0.017 0.017 0.012
1 3.7 1.5 7.1
Results are from duplicating experiments. The resistant fold is the IC50 ratio of mutant/WT and present as mean SD. WT, wild-type; BCP, basal core promoter; PC, precore.
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Cui et al.
Fig. 2. Sensitivity of wild-type and mutant-type HBV A, B, or C genotypes to antiviral NA drugs. Replicating capacity of wild-type and mutant-type HBV in various concentrations of antiviral drugs was determined by Southern blotting and the density of signal were normalized to that of the untreated strain of either wild-type or mutant-type HBV. The data were used to determine IC50s by a nonlinear regression method. WT, wild type; BCP, basal core promoter; PC, precore.
J. Med. Virol. DOI 10.1002/jmv
BCP, PC on HBV Replication and Susceptibility to NAs
The present study also evaluated the effect of BCP and PC mutation on the sensitivity of the various HBV genotypes to NAs. IC50s for lamivudine, adefovir, entecavir, and tenofovir were higher in BCP and/ or PC-mutant HBV strains compared with wild-type HBV. This finding suggests that a higher concentration or more potent antiviral agent might be needed to suppress the viral replication of these mutant strains effectively. Several studies reported different response to antiviral drugs according to HBV genotype. Although HBV genotype C has a poor response to interferonbased therapy compared with genotypes A or B [Kao et al., 2000; Wai et al., 2002; Flink et al., 2006], HBV genotypes have little different effect on treatment with NAs [Yuen et al., 2003; Wiegand et al., 2008]. This study showed conspicuously different susceptibility to entecavir and tenofovir. For example, in genotype C with a PC mutant, the IC50s for entecavir and tenofovir were 10.7- and 1.5-fold those for the wild type. By contrast, in genotype B with a PC mutant, the IC50s for entecavir and tenofovir were 5and 10.2-fold of those for the wild type. This finding suggests that HBV genotype C with a PC mutant is more susceptible to tenofovir than entecavir. However, HBV genotype B with a PC mutant was found to be more susceptible to entecavir. Therefore, clinical studies may clarify this issue. In conclusion, BCP and PC mutations increased the viral replication competence regardless of HBV genotype in vitro. The HBV with a BCP mutation affects different replication competency depending on HBV genotype. However, HBVs with a PC mutation show similar replication competence regardless of genotype. REFERENCES Akarca US, Greene S, Lok AS. 1994. Detection of precore hepatitis B virus mutants in asymptomatic HBsAg-positive family members. Hepatology 19:1366–1370. Aritomi T, Yatsuhashi H, Fujino T, Yamasaki K, Inoue O, Koga M, Kato Y, Yano M. 1998. Association of mutations in the core promoter and precore region of hepatitis virus with fulminant and severe acute hepatitis in Japan. J Gastroenterol Hepatol 13:1125–1132. Baptista M, Kramvis A, Kew MC. 1999. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 29:946–953. Baumert TF, Rogers SA, Hasegawa K, Liang TJ. 1996. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J Clin Invest 98:2268–2276. Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. 1996. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 70:5845–5851. Carman WF, Fagan EA, Hadziyannis S, Karayiannis P, Tassopoulos NC, Williams R, Thomas HC. 1991. Association of a precore genomic variant of hepatitis B virus with fulminant hepatitis. Hepatology 14:219–222. Chan HL, Hussain M, Lok AS. 1999. Different hepatitis B virus genotypes are associated with different mutations in the core promoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 29:976–984.
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