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J Clin Invest. Feb 15, 2001; 107(4): 449–455.
PMCID: PMC199250
The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance
Suzane Kioko Ono,1,2 Naoya Kato,1 Yasushi Shiratori,1 Jun Kato,1 Tadashi Goto,1 Raymond F. Schinazi,3 Flair José Carrilho,2 and Masao Omata1
1Department of Gastroenterology, Faculty of Medicine, University of Tokyo, Tokyo, Japan2Department of Gastroenterology, School of Medicine, University of Sao Paulo, Sao Paulo, Brazil3Veterans Affairs Medical Center and Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
Address correspondence to: Naoya Kato, Department of Gastroenterology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Phone: 81-3-3815-5411 ext. 33056; Fax: 81-3-3814-0021; E-mail: kato-2im/at/h.u-tokyo.ac.jp.
Received August 17, 2000; Accepted January 15, 2001.
After receiving lamivudine for 3 years to treat chronic hepatitis B, 67–75% of patients develop B-domain L528M, C-domain M552I, or M552V mutations in the HBV polymerase that render hepatitis B virus (HBV) drug-resistant. The aim of this study was to evaluate the influence of these mutations on viral replication and resistance to antiviral agents. We investigated the replication fitness and susceptibility of the wild-type and five mutant HBVs (L528M, M552I, M552V, L528M/M552I, and L528M/M552V) to 11 compounds [lamivudine, adefovir, entecavir (BMS-200475) (+)-BCH-189 (±)-FTC (racivir) (–)-FTC (emtricitabine) (+)-FTC, L-D4FC, L-FMAU (clevudine), D-DAPD, and (–)-carbovir] by transfecting HBV DNA into hepatoma cells and monitoring viral products by Southern blotting. The replication competency of the single C-domain mutants M552I and M552V was markedly decreased compared with that of wild-type HBV. However, addition of the B-domain mutation L528M restored replication competence. Only adefovir and entecavir were effective against all five HBV mutants, and higher doses of these compounds were necessary to inhibit the double mutants compared with the single mutants. The B-domain mutation (L528M) of HBV polymerase not only restores the replication competence of C-domain mutants, but also increases resistance to nucleoside analogues.
Despite the availability for almost 20 years of safe and effective vaccines against hepatitis B, chronic infection with hepatitis B virus (HBV) remains among the ten most common causes of death worldwide (1). Recently, lamivudine [(–)-β-L-2′,3′-dideoxy-3′-thiacytidine (3TC)] became the first approved oral therapy for the treatment of HBV (2). Clinical data have shown that lamivudine treatment rapidly reduces the levels of HBV DNA, is well tolerated, and improves liver histology (3, 4). However, a defined course of 52 weeks’ treatment provides a sustained response rate of 17–33% (loss of HBeAg). Discontinuation of therapy at 52 weeks is followed by relapse in patients who do not lose HBeAg by that point, and these patients may benefit from long-term therapy (5, 6). However, prolonged use of lamivudine therapy has been associated with increased emergence of lamivudine-resistant HBV with amino-acid substitutions in the B domain (L528M) and in the YMDD motif of the C domain (M552I and M552V) of the viral DNA polymerase (714). The emergence rate of lamivudine-resistant HBV ranges from 17–46% at 1 year to as high as 67–75% after 3–4 years of continuous therapy (4, 6, 15).
The YMDD motif, a conserved motif in RNA-dependent DNA polymerase, is involved in nucleotide binding in the catalytic site of the polymerase (16, 17). It was previously demonstrated that owing to replication competence and lamivudine sensitivity, viruses having M552I or M552V sequences may appear during treatment with lamivudine (18, 19). On the other hand, the B domain of DNA polymerase is an element responsible for template positioning (20). Amino acid substitution in the B domain (L528M) of HBV polymerase was described in patients receiving lamivudine, accompanying the M552I or M552V mutation, and in patients receiving famciclovir without any mutation in the YMDD motif (10, 21).
Liaw et al. recently reported that exacerbation occurred in 41% of patients after the emergence of YMDD motif mutations during continued use of lamivudine (22). In addition, lamivudine-resistant HBV is associated with advanced hepatic fibrosis and severe microinflammatory changes in patients with recurrent HBV infection after orthotopic liver transplantation (23). Lamivudine-resistant HBV also can cause severe hepatitis in patients coinfected with HIV and HBV (24). Thus, the need to develop new antivirals and new strategies to treat HBV infections is becoming clear. Previously, we demonstrated that adefovir decreased replication of wild-type and lamivudine-resistant HBV (25); however, new antivirals are becoming available, and there is a pressing need for further studies to determine their potential as single agents and for combination chemotherapy.
The aim of this study was to evaluate the replication competence and susceptibility of wild-type HBV and five different mutants (L528M, M552I, M552V, L528M/M552I, and L528M/M552V) to 11 drugs [lamivudine; 9-(2-bis [pivaloyloxymethyl] methoxyethyl) adenine (adefovir); [1S-(1α,3 α,4 β)]-2-amino-1,9-dihydro-9[4-hydroxy-3-(hydroxymethyl)-2-methylenecyclopentyl]-6H-purin-6-one (entecavir); (+)-β-2′,3′-dideoxy-3′-thiacytidine (BCH-189) (the plus enantiomer of lamivudine); (±)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (FTC) (racivir); (–)-FTC (emtricitabine, coviracil); (+)-FTC (the plus enantiomer of emtricitabine); (–)-β-L-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (L-D4FC); 2′-fluoro-5-methyl-β-L-arabinofuranosyluracil (L-FMAU, clevudine); (–)-β-D-2,6-diaminopurine dioxolane (DAPD); and (–)-carbovir].
Chemicals.
Lamivudine was generously donated by Glaxo-Wellcome (Middlesex, United Kingdom). Adefovir was a gift from Gilead Sciences (Foster City, California, USA). Entecavir (BMS-200475), a guanosine analogue, was synthesized by the method of Bisacchi et al. (26). The other antiviral agents were synthesized in R.F. Schinazi’s laboratory. All compounds were coded and remained coded until the preliminary results were obtained.
Cells.
HuH-7 cells (Human Science Research Resource Bank, Osaka, Japan) (27) were cultured in DMEM (Life Technologies Inc., Rockville, Maryland, USA) supplemented with 10% FBS. The 2.2.15 cells (clonal cells derived from HepG2 cells that were transfected with a plasmid containing HBV DNA) that secrete HBV virions were kindly provided by G. Acs (Mount Sinai Medical Center, New York, New York, USA) (28). The 2.2.15 cells were maintained in DMEM supplemented with 20% FBS.
HBV plasmids (wild-type, “single” and “double” mutants, and dimer).
Wild-type HBV DNA, extracted from the serum of a 54 year-old Japanese man with HBeAg-positive (subtype ayw, genotype D) cirrhosis, was amplified and cloned as described previously (18, 25). HBV DNA was extracted from 100 μl serum using a SepaGene kit (Sanko Junyaku, Tokyo, Japan) according to the manufacturer’s instructions. Five mutants were prepared. First, three single mutants (L528M, M552I, and M552V) were made by substituting nucleotides in order to change the codon for Met in the YMDD motif to Ile (M552I) or Val (M552V) or a codon for Leu in the B domain to Met (L528M) using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, California, USA). Similarly, two double mutants (L528M/M552I and L528M/M552V) were constructed by adding L528M substitution to the C-domain single mutants M552I and M552V. To confirm the introduction of mutations, the polymerase genes of the mutants were sequenced using a cycle DNA sequencing system (Perkin-Elmer Applied Biosystems, Foster City, California, USA) as described previously (29).
Plasmid pSM2 containing a head-to-tail dimer of HBV was kindly provided by S. Günther (Heinrich-Pette-Institut fur Experiementelle Virologie, Hamburg, Germany) (30).
Transfection of HBV DNA into HuH-7 cells.
HuH-7 cells at 80–90% confluence (in 60-mm dishes) were transfected with 0.9 μg full-length HBV DNA wild-type, mutants, or pSM2 using Effectene transfection reagent (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Twenty-four hours after transfection, the medium was changed and reincubated with drug-free medium or medium containing 0.0001, 0.001, 0.01, 0.1, 1, or 10 μM of each compound. Medium and cells (rinsed three times with cold PBS) were harvested 3 days later. The efficiency of transfection was monitored by cotransfecting 0.1 μg β-galactosidase expression plasmid, pCMV β (CLONTECH Laboratories Inc., California, USA). Assays for β-galactosidase in extracts of HuH-7 cells were performed as described previously (25).
The medium of 2.2.15 cells at 80–90% confluence (in 60-mm dishes) was changed and reincubated with drug-free medium or medium containing 0.0001, 0.001, 0.01, 0.1, 1, or 10 μM of lamivudine or entecavir. Medium and cells (rinsed three times with cold PBS) were harvested 3 days later.
Experiments were performed at least in duplicate.
Isolation of core-particle–related HBV DNA.
Purification of HBV DNA from intracellular core particles was accomplished using the method described by Günther et al. (30) with minor modifications. Briefly, cells were suspended in 500 μl of lysis buffer containing 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1% NP-40, transferred to an Eppendorf tube, vortexed, and allowed to stand on ice for 15 minutes. Nuclei were pelleted by centrifugation at 4°C, 15,000 g for 1 minute. The supernatant was transferred to a new tube, adjusted to 10 mM MgCl2, and digested with 100 μg/ml of DNase I for 30 minutes at 37°C. To stop the reaction, EDTA was added to a final concentration of 25 mM. Then, 0.5 mg/ml proteinase K and 1% sodium dodecylsulfate were added and incubated at 50°C for 4 hours. Phenol-chloroform (1:1) extraction was performed, and then the nucleic acids were ethanol precipitated along with a glycogen carrier.
Southern blot hybridization of HBV DNA.
HBV DNA was resolved in 1.5% agarose gels, transferred to nylon membranes (Hybond N+; Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) by Southern blotting, and hybridized with an alkaline-phosphatase–labeled wild-type full-length HBV DNA probe generated with the Gene Images AlkPhos Direct labeling system (Amersham Pharmacia Biotech). Chemiluminescent detection was performed with CDP-Star (Amersham Pharmacia Biotech) and analyzed using an LAS1000 image analyzer (Fuji Photo Film, Tokyo, Japan).
Drug susceptibility analysis (determination of EC50) by measuring single-stranded HBV DNA.
To compare the effect of the 11 antiviral agents on the wild-type HBV and five mutants, HuH-7 cells were transfected with HBV DNA, and the antivirals were added at a concentration of 10 μM. For those antivirals that inhibited the replication of HBV by more than 50% at this concentration, increasing concentrations (0.0001 to 10 μM) of the compound were applied to calculate the effective concentration required to reduce HBV replication by 50% (EC50). To evaluate the susceptibility of HBV to the antiviral agents, Southern blot hybridization of DNA extracts from transfected cells was performed. The single-stranded HBV DNA band, previously shown to represent HBV intermediates (30, 31), was analyzed to assess the efficacy of the RT inhibitors on HBV replication. This single-stranded band was quantified and normalized for transfection efficiency based on β-galactosidase activity. Data are shown as the mean ± SD of at least two experiments.
Replication competency of the five HBV mutants.
To evaluate the effect of mutations in the polymerase gene on HBV replication, the replication ability of the wild-type HBV and five mutants (L528M, M552I, M552V, L528M/M552I, and L528M/M552V) was examined in a transient transfection cell culture assay system. Southern blot hybridization of DNA extracts showed the presence of a single-stranded band (representative of HBV replication intermediates) for each construct, indicating that they were replication-competent (Figure (Figure1).1). These bands were quantified, adjusted for the efficiency of transfection according to the β-galactosidase assay of cotransfected pCMVβ, and determined by taking the single-stranded band of the wild type as 100%. Although the single B-domain mutation L528M did not affect replication ability, the single C-domain mutations M552I and M552V had markedly decreased replication ability compared with the wild-type (14% and 10% of the wild-type HBV, respectively) (Figure (Figure1).1). In contrast, the double mutants L528M/M552I and L528M/M552V had better replication ability (55% and 68% of the wild-type HBV, respectively) when compared with the single C-domain mutants M552I and M552V, the double mutant replicating 3.9 times more than the M552I mutant and 6.8 times more than the M552V mutant.
Figure 1
Figure 1
Southern blot hybridization analysis of replication of wild-type HBV and five mutants. Lanes correspond to DNA extracted from viral core particles derived from HuH-7 cells that were transfected with DNA of wild-type HBV or one of five mutants. Single-stranded (more ...)
Effect of antiviral agents against wild-type HBV.
First, to assess the effect of the 11 compounds on wild-type HBV replication in vitro, HuH-7 cells transfected with wild-type HBV DNA were incubated with 10 μM of each compound. Southern blot hybridization of DNA extracts showed the presence of a single-stranded band in the drug-free samples. All compounds except (+)-BCH-189 decreased the single-stranded band of wild-type HBV, showing that wild-type HBV is susceptible to the majority of the compounds tested (Table (Table1).1). Seven compounds [lamivudine, adefovir, entecavir (±)-FTC (–)-FTC, L-D4FC, and L-FMAU] inhibited the replication of wild-type HBV more than 50% at a concentration of 10 μM. Therefore, the EC50 of these seven drugs was determined against wild-type HBV using increasing concentrations (0.0001, 0.001, 0.01, 0.1, 1, and 10 μM) by performing at least two independent experiments (Figure (Figure22 and Table Table1).1). Entecavir, L-D4FC, L-FMAU (–)-FTC, lamivudine, adefovir, and (±)-FTC (in relative order of potency) were “effective” (EC50 < 10 μM) against wild-type HBV with an EC50 of 0.00036, 0.033, 0.053, 0.24, 0.56, 0.58, and 1.85 μM, respectively (Tables (Tables11 and and2).2). Entecavir, L-D4FC, L-FMAU, and (–)-FTC inhibited wild-type HBV replication 1,556, 17, 11, and two times more efficiently than did lamivudine, respectively.
Table 1
Table 1
EC50 values of compounds and level of replication of wild-type and five mutants HBV at 10 μM concentration treatment
Figure 2
Figure 2
Representative Southern blot hybridization used to determine the EC50 value of entecavir against wild-type HBV. (a) Southern blot hybridization analysis of replication of the wild-type treated with entecavir. Lanes correspond to DNA extracted from viral (more ...)
Table 2
Table 2
Comparison of potencies of compounds against wild-type and lamivudine-resistant mutant HBV
Comparison of the effect of lamivudine and entecavir against wild-type HBV in pSM2-transfected HuH7 cells and 2.2.15 cells.
The potency of lamivudine and entecavir against wild-type HBV was also validated in pSM2-transfected HuH7 cells and 2.2.15 cells. The EC50 values of lamivudine and entecavir were 0.19 and 0.0008 μM, respectively, in pSM2-transfected HuH7 cells; and 0.55 and 0.00025 μM, respectively, in 2.2.15 cells (Figure (Figure3).3). Entecavir inhibited wild-type HBV replication 2,200 times more strongly than did lamivudine in pSM2-transfected HuH7 cells, and 237 times more strongly in 2.2.15 cells.
Figure 3
Figure 3
Susceptibility of HBV dimer (pSM2) (a) and HBV from 2.2.15 cells (b) to lamivudine and entecavir: drug inhibition curves of wild-type HBV transfected into HuH-7 cells treated with the indicated concentrations of lamivudine and entecavir.
Effect of antiviral agents on three single mutant HBVs.
To analyze the in vitro antiviral effect of the compounds on three single mutant HBVs, HuH-7 cells transfected with L528M, M552I, or M552V were incubated with 10 μM of each compound. Adefovir, entecavir (±)-FTC (–)-FTC, L-D4FC, and L-FMAU inhibited replication of all three single mutant HBVs more than 50% at a concentration of 10 μM (Table (Table1).1). Therefore, we determined the EC50 of these six drugs against the three single mutants using increasing concentrations (0.0001–10 μM) of the compounds by performing at least two independent experiments (Table (Table1).1). (±)-FTC and (–)-FTC were effective only against single B-domain mutant L528M, whereas L-D4FC and L-FMAU were effective against both single B-domain mutant L528M and C-domain mutant M552V (Tables (Tables11 and and2).2). However, only adefovir and entecavir were effective against all three single mutants: L528M, M552I, and M552V (Tables (Tables11 and and2).2). Although adefovir and entecavir were effective against all three single mutants, the EC50 values of adefovir and entecavir for the three single mutants were up to 8.6 and 166 times higher than for wild-type HBV, respectively.
Effect of antiviral agents against two double mutants HBV.
To analyze the in vitro antiviral effect of the previously mentioned 11 compounds on two double HBV mutants, HuH-7 cells transfected with L528M/M552I or L528M/M552V were incubated with 10 μM of each compound. Only adefovir and entecavir inhibited replication of these two double mutant HBVs more than 50% at a concentration of 10 μM (Table (Table1).1). The EC50 values of adefovir and entecavir against the two double mutant HBVs were determined using increasing concentrations of the compounds (0.0001 to 10 μM), by performing at least two independent experiments (Tables (Tables11 and and2).2). The EC50 values of adefovir for the mutants were four to 16 times higher than those for wild-type HBV and the EC50 values of entecavir for the mutants were 694 to 778 times higher than those for wild-type HBV. Although the single C-domain mutants M552I and M552V were less susceptible to entecavir than were wild-type HBV, the addition of the B-domain L528M mutation to the C-domain mutants (double mutants L528M/M552I and L528M/M552V) resulted in HBV that was more resistant to entecavir.
Although lamivudine was approved for the treatment of patients with chronic hepatitis B, short-term treatment is usually insufficient to clear the virus. Moreover, long-term treatment is associated with the development of drug-resistant HBV in 14–75% of patients (2, 4, 15, 32). These lamivudine-resistant HBV harbor M552I or M552V mutations in the C domain of the polymerase gene. The L528M mutation frequently accompanies M552V and has recently been reported to accompany M552I occasionally (710, 12). To summarize three published reports, the clinical frequency of lamivudine-resistant mutants was 18.6% for M552I, 1.4% for M552V, 11.4% for L528M/M552I, and 64.3% for L528M/M552V (10, 11, 13).
Given that mutations in the polymerase gene have been associated with changes in the replication competency of the virus (18, 19), we examined the influence, singly or in combination, of the B-domain mutation (L528M) and the two C-domain mutations (M552I and M552V) on replication ability. Compared with the wild-type HBV, the single C-domain mutants M552I and M552V had markedly decreased replication abilities (18). Of particular interest, the double mutants with both B- and C-domain mutations (L528M/M552I and L528M/M552V) replicated significantly better than did the single C-domain mutants, suggesting that the B-domain mutation L528M rescued the defective replication competence of the C-domain mutants. It was previously indicated that L528M could compensate for the impact of a mutation in the YMDD motif on viral replication in vitro (33). The B-domain L528 in the amino acid 521-537 helix is close enough to interact with M552 of the YMDD loop in a hypothetical molecular model of HBV RT (10). It could be speculated that the L528M mutation reduces the imbalance of conformation caused by the M552V and M552I mutations, thereby improving the replication competence of single C-domain mutants.
Of the 11 compounds tested, entecavir, L-D4FC, L-FMAU (–)-FTC, lamivudine, and adefovir inhibited the replication of wild-type HBV effectively, with an EC50 less than 1 μM. These results are comparable with previous reports that demonstrated inhibition of HBV replication by 50% at concentrations of less than 0.1 μM in 2.2.15 cells (3438). Because several different groups tested these compounds in different ways, it was difficult to compare their EC50 values, as they varied according to the assay used, cell type used, marker of viral replication selected, composition of the medium, and time in culture (39). We studied the effect of the 11 compounds simultaneously. Therefore, we could determine and compare their relative potencies. Entecavir, L-D4FC, L-FMAU, and (–)-FTC inhibited wild-type HBV replication two to 1,556 times more than lamivudine. Similar results were obtained in 2.2.15 cells and pSM2-transfected HuH7 cells. In these cells, entecavir was 237–2,200 times more potent than lamivudine. In general, the more potent the antiviral agents used to suppress viral replication, the less likely the virus is to develop drug-resistant mutations, because mutations arise as replication errors (4043). Therefore, in the absence of toxicological considerations of these experimental agents, entecavir, L-D4FC, and L-FMAU are potentially useful “first line” drugs for the treatment of HBV. However, it must be noted that in vitro sensitivities do not always match the sensitivities of virus infection in humans in vivo. Therefore, the ultimate test is their effectiveness in patients with chronic hepatitis B.
While adefovir, entecavir (+)-FTC (–)-FTC, L-D4FC, and L-FMAU are effective (EC50 < 10 μM) against some of the five lamivudine-resistant HBV mutants, only adefovir and entecavir are effective against all five mutants. As previously described, adefovir might be a good treatment option in those patients who have failed lamivudine therapy because of drug-resistant HBV (25). In fact, it was recently demonstrated that adefovir resulted in a rapid and sustained reduction in HBV DNA levels associated with improvement in liver function in patients who failed to respond to lamivudine therapy (44). In addition, based on our data, entecavir could be an option for the treatment of lamivudine-resistant mutants. However, it must be noted that the EC50 values for lamivudine-resistant mutants were 2 to 778 times higher than those for the wild-type HBV. Therefore, the dose of entecavir necessary to treat lamivudine-resistant mutants would be considerably higher than that used for wild-type HBV.
L-D4FC and L-FMAU were effective against single B- or C-domain mutants L528M and M552V with an EC50 of less than 2 μM, but were ineffective against the double mutant L528M/M552V, with an EC50 exceeding 10 μM. In addition, the EC50 of entecavir for the double mutants was about 500 times higher than that for the single B-domain mutant L528M, and 90 times higher than that for the single C-domain mutants. The addition of the B-domain mutation L528M to C-domain mutants may contribute to increased levels of resistance to entecavir, L-D4FC, and L-FMAU but does not seem to increase the level of resistance to adefovir, suggesting that the resistance pattern for adefovir is unique and different from those of entecavir, L-D4FC, and L-FMAU.
Although further studies are needed to address the cytotoxicity of these drugs in humans, the doses of the compounds used in our study are far below the toxic doses reported by others. Using 2.2.15 cells, no apparent cytotoxicity was noted at concentrations greater than 1,000, 150, and 30 μM for lamivudine, adefovir, and entecavir, respectively (34, 45, 46). For L-D4FC, the concentration required to inhibit 50% of HepG2 growth was estimated to be 20 μM (35). Unfortunately, L-D4FC is cytotoxic in various cells and with prolonged treatment has been shown to increase lactic acid production in HepG2 (47). L-FMAU (±)-FTC, and (–)-FTC did not show any cytotoxicity up to 200 μM in 2.2.15 cells (3638). In addition, with the exception of entecavir, in vivo studies have shown that these compounds are not associated with overt toxicity at high doses (>100 mg/kg). In duck hepatitis B virus–infected ducklings, treatment with 15–30 mg/kg of adefovir or 40 mg/kg of L-FMAU produced no toxic side effects (46, 48). In the woodchuck model, 0.5–0.1 mg/kg of entecavir, 1–4 mg/kg of L-D4FC, or 20–30 mg/kg of (–)-FTC inhibited replication of woodchuck hepatitis virus without associated toxicity (4952).
As learned from the treatment of HIV, it is likely that combinations of HBV drugs should be used to maximize suppression of replication and consequently decrease the probability of the emergence of a drug-resistant virus (40, 53, 54). This approach would permit the use of lower doses of the antiviral agents and, therefore, reduce the likelihood of side effects. It also seems advantageous to combine adefovir with lamivudine, entecavir, L-D4FC, L-FMAU, or (–)-FTC in an effort to better suppress HBV replication and delay the development of resistance. Although entecavir, L-D4FC, and L-FMAU had the lowest EC50, a combination of these three drugs may be compromised, as they have a similar cross-resistance profile. Further studies are necessary to determine the potential synergistic interaction of compounds in combination therapy.
Acknowledgments
This study was supported by a Research Grant for Immunology, Allergy, and Organ Transplantation from the Ministry of Health and Welfare, Japan. S.K. Ono is a Research Fellow of the Japan Society for the Promotion of Science. R.F. Schinazi is supported in part by the Department of Veterans Affairs and NIH grant RO1-AI-41980. We thank Mitsuko Tsubouchi for technical assistance. Neither this study nor the authors received grant support from the pharmaceutical companies whose products are examined.
1. World Health Organization warns of growing “crisis of suffering.” http://www.who.ch/whr/1997/presse.htm. [PubMed]
2. Gordon D, Walsh JH. Hepatitis drugs win approval. Gastroenterology. 1998; 116:235–236.
3. Honkoop P, de Man RA, Zondervan PE, Schalm SW. Histological improvement in patients with chronic hepatitis B virus infection treated with lamivudine. Liver. 1997; 17:103–106. [PubMed]
4. Lai CL, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med. 1998; 339:61–68. [PubMed]
5. Omata M. Treatment of chronic hepatitis B infection. N Engl J Med. 1998; 339:114–115. [PubMed]
6. Lai CL. Antiviral therapy for hepatitis B and C in Asians. J Gastroenterol Hepatol. 1999; 14(Suppl.):S19–S21. [PubMed]
7. Bartholomew MM, et al. Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet. 1997; 349:20–22. [PubMed]
8. Ling R, et al. Selection of mutations in the hepatitis B virus polymerase during therapy of transplant recipients with lamivudine. Hepatology. 1996; 24:711–713. [PubMed]
9. Tipples GA, et al. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology. 1996; 24:714–717. [PubMed]
10. Allen MI, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology. 1998; 27:1670–1677. [PubMed]
11. Chayama K, et al. Emergence and takeover of YMDD motif mutant hepatitis B virus during long-term lamivudine therapy and re-takeover by wild type after cessation of therapy. Hepatology. 1998; 27:1711–1716. [PubMed]
12. Yeh CT, Chien RN, Chu CM, Liaw YF. Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy. Hepatology. 2000; 31:1318–1326. [PubMed]
13. Benhamou Y, et al. Long-term incidence of hepatitis B virus resistance to lamivudine in human immunodeficiency virus-infected patients. Hepatology. 1999; 30:1302–1306. [PubMed]
14. Bartholomeusz A, Schinazi RF, Locarnini SA. Significance of mutations in the hepatitis B virus polymerase selected by nucleoside analogues and implications for controlling chronic disease. Viral Hepatitis Reviews. 1998; 4:167–187.
15. Lau DT-Y, et al. Long-term lamivudine therapy for chronic hepatitis B. Antivir Ther. 2000; 5(Suppl. 1):43. (Abstr.)
16. Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. EMBO J. 1989; 8:3867–3874. [PubMed]
17. Kamer G, Argos P. Primary structural comparison of RNA-dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 1984; 12:7269–7282. [PMC free article] [PubMed]
18. Ono-Nita SK, et al. YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: a study by in vitro full-length viral DNA transfection. Hepatology. 1999; 29:939–945. [PubMed]
19. Melegari M, Scaglioni PP, Wands JR. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology. 1998; 27:628–633. [PubMed]
20. Jacobo-Molina A, et al. Crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double-stranded DNA at 3.0 A resolution shows bent DNA. Proc Natl Acad Sci USA. 1993; 90:6320–6324. [PubMed]
21. Seigneres B, et al. Evolution of hepatitis B virus polymerase gene sequence during famciclovir therapy for chronic hepatitis B. J Infect Dis. 2000; 181:1221–1233. [PubMed]
22. Liaw YF, Chien RN, Yeh CT, Tsai SL, Chu CM. Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy. Hepatology. 1999; 30:567–572. [PubMed]
23. Ben-Ari Z, Pappo O, Zemel R, Mor E, Tur-Kaspa R. Association of lamivudine resistance in recurrent hepatitis B after liver transplantation with advanced hepatic fibrosis. Transplantation. 1999; 68:232–236. [PubMed]
24. Bessesen M, Ives D, Condreay L, Lawrence S, Sherman KE. Chronic active hepatitis B exacerbations in human immunodeficiency virus-infected patients following development of resistance to or withdrawal of lamivudine. Clin Infect Dis. 1999; 28:1032–1035. [PubMed]
25. Ono-Nita SK, et al. Susceptibility of lamivudine-resistant hepatitis B virus to other reverse transcriptase inhibitors. J Clin Invest. 1999; 103:1635–1640. [PMC free article] [PubMed]
26. Merchant Z, et al. BMS-200475, a novel carbocyclic 2′-deoxyguanosine analog with potent and selective anti-hepatitis B virus activity in vitro. Bioorg Med Chem Lett. 1997; 7:127–132.
27. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982; 42:3858–3863. [PubMed]
28. Sells MA, Chen ML, Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci USA. 1987; 84:1005–1009. [PubMed]
29. Togo G, et al. A transforming growth factor beta type II receptor gene mutation common in sporadic cecum cancer with microsatellite instability. Cancer Res. 1996; 56:5620–5623. [PubMed]
30. Günther S, et al. 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. 1995; 69:5437–5444. [PMC free article] [PubMed]
31. Yokosuka O, Omata M, Imazeki F, Okuda K, Summers J. Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA. Hepatology. 1985; 5:728–734. [PubMed]
32. Dienstag JL, et al. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med. 1999; 341:1256–1263. [PubMed]
33. Fu L, Cheng YC. Role of additional mutations outside the YMDD motif of hepatitis B virus polymerase in L(-)SddC (3TC) resistance. Biochem Pharmacol. 1998; 55:1567–1572. [PubMed]
34. Innaimo SF, et al. Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus. Antimicrob Agents Chemother. 1997; 41:1444–1448. [PMC free article] [PubMed]
35. Zhu YL, Dutschman DE, Liu SH, Bridges EG, Cheng YC. Anti-hepatitis B virus activity and metabolism of 2′,3′-dideoxy-2′,3′-didehydro-beta-L(-)-5-fluorocytidine. Antimicrob Agents Chemother. 1998; 42:1805–1810. [PMC free article] [PubMed]
36. Balakrishna Pai S, Liu SH, Zhu YL, Chu CK, Cheng YC. Inhibition of hepatitis B virus by a novel L-nucleoside, 2′-fluoro-5-methyl-beta-L-arabinofuranosyl uracil. Antimicrob Agents Chemother. 1996; 40:380–386. [PMC free article] [PubMed]
37. Jansen RW, Johnson LC, Averett DR. High-capacity in vitro assessment of anti-hepatitis B virus compound selectivity by a virion-specific polymerase chain reaction assay. Antimicrob Agents Chemother. 1993; 37:441–447. [PMC free article] [PubMed]
38. Furman PA, et al. The anti-hepatitis B virus activities, cytotoxicities, and anabolic profiles of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine. Antimicrob Agents Chemother. 1992; 36:2686–2692. [PMC free article] [PubMed]
39. Hirsch MS, et al. Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. International AIDS Society—USA Panel. JAMA. 1998; 279:1984–1991. [PubMed]
40. Condra JH. Resisting resistance: maximizing the durability of antiretroviral therapy. Ann Intern Med. 1998; 128:951–954. [PubMed]
41. Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science. 1995; 267:483–489. [PubMed]
42. Tsiang M, Rooney JF, Toole JJ, Gibbs CS. Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy. Hepatology. 1999; 29:1863–1869. [PubMed]
43. Havlir DV, Richman DD. Viral dynamics of HIV: implications for drug development and therapeutic strategies. Ann Intern Med. 1996; 124:984–994. [PubMed]
44. Peters M, et al. Adefovir dipivoxil treatment of hepatitis B virus disease in patients failing lamivudine therapy. Antivir Ther. 2000; 5(Suppl. 1):45.
45. Kruining J, Heijtink RA, Schalm SW. Antiviral agents in hepatitis B virus transfected cell lines: inhibitory and cytotoxic effect related to time of treatment. J Hepatol. 1995; 22:263–267. [PubMed]
46. Heijtink RA, et al. Inhibitory effect of 9-(2-phosphonylmethoxyethyl)-adenine (PMEA) on human and duck hepatitis B virus infection. Antiviral Res. 1993; 21:141–153. [PubMed]
47. Shi J, et al. Synthesis and biological evaluation of 2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (D4FC) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase. J Med Chem. 1999; 42:859–867. [PubMed]
48. Aguesse-Germon S, et al. Inhibitory effect of 2′-fluoro-5-methyl-beta-L-arabinofuranosyl-uracil on duck hepatitis B virus replication. Antimicrob Agents Chemother. 1998; 42:369–376. [PMC free article] [PubMed]
49. Genovesi EV, et al. Efficacy of the carbocyclic 2′-deoxyguanosine nucleoside BMS-200475 in the woodchuck model of hepatitis B virus infection [erratum 1999, 43:726] Antimicrob Agents Chemother. 1998; 42:3209–3217. [PMC free article] [PubMed]
50. Le Guerhier F, et al. 2′,3′-dideoxy-2′,3′-didehydro-b-L-5-fluorocytidine (b-L-FD4C) exhibits a more potent antiviral effect than lamivudine in chronically WHV infected woodchucks. Hepatology. 1999; 30:344A. (Abstr.)
51. Cullen JM, et al. In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks. Antimicrob Agents Chemother. 1997; 41:2076–2082. [PMC free article] [PubMed]
52. Korba BE, Schinazi RF, Cote P, Tennant BC, Gerin JL. Effect of oral administration of emtricitabine on woodchuck hepatitis virus replication in chronically infected woodchucks. Antimicrob Agents Chemother. 2000; 44:1757–1760. [PMC free article] [PubMed]
53. Balzarini J. Suppression of resistance to drugs targeted to human immunodeficiency virus reverse transcriptase by combination therapy. Biochem Pharmacol. 1999; 58:1–27. [PubMed]
54. Schinazi, R.F. 1991. Combined therapeutic modalities for viral infections: rationale and clinical potential. In Synergism and antagonism in chemotherapy. T.-C. Chou and D.C. Rideout, editors. Academic Press. Orlando, Florida, USA. 110–181.
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