Hepatitis B virus (HBV) infection is among the top 10 viral infections, affecting an estimated 300 million people worldwide and over 1.5 million in the United States alone (10
). Chronic HBV infection can lead to cirrhosis, hepatocellular carcinoma, and liver failure. Treatment of chronically HBV-infected patients with alpha interferon (28
) is limited by side effects, incomplete efficacy, restriction to patients with compensated disease, and the requirement for parenteral administration (8
). HBV, a hepadnavirus, replicates through an intermediate reverse transcription step carried out by the viral polymerase (19
), which is functionally and structurally related to human immunodeficiency virus (HIV) reverse transcriptase (RT). Some of the nucleoside analogs developed to treat HIV infection are highly potent against HBV infection (4
) at concentrations below cytotoxic thresholds. Treatment of chronically HBV-infected patients with nucleoside or nucleotide analogs (Fig. ), like lamivudine (3TC), emtricitabine (FTC), famciclovir (the prodrug of penciclovir [PCV]), adefovir dipivoxil (ADV [also called PMEA]), and lobucavir (LBV), leads to significant decreases in serum virus levels (26
). Treatment with the nucleoside or nucleotide analogs has shown immediate clinical benefits such as reduced viral load, suppression of progression of liver disease, and induction of immunological clearance or seroconversion (6
). Drug-resistant strains of HBV containing specific polymerase mutations emerge upon prolonged 3TC treatment (14
; H. Fontaine, V. Thiers, and S. Pol, Letter, Ann. Intern. Med. 131:
716–717, 1999) and are the primary cause of treatment failure. Treatment of HBV-infected patients with 3TC in phase III clinical studies showed a sequential increase in appearance of genotypic resistance in HBV patients: 24% in the first year, 42% in the second year, 52% in the third year, and 67% in the fourth year (N. W. Y. Leung, C. L. Lai, J. Dienstag, G. Schiff, J. Heathcote, M. Atkins, C. Marr, and W. C. Maddrey, presented at the Management of Hepatitis B Meeting, 8 to 10 September 2000).
Chemical structures of dCTP, 3TCTP, FTCTP, ADVDP, LBVTP, and PCVTP.
As with other nucleotide polymerases, the triphosphates of the nucleotide substrates or their analog inhibitors are the catalytically active forms for polymerization by HBV polymerase, and the polymerization reaction has been shown to be Mg2+
ion dependent (34
). Two of three catalytically essential aspartic acid residues are part of the highly conserved YMDD motif at the active site of HBV polymerase and its close viral relatives, including HIV type 1 (HIV-1) RT. The most common 3TC resistance mutations, Met552Ile and Met552Val (Met552Ile/Val), appear at the Met (M) position in the YMDD motif of the HBV polymerase, analogous to the lamivudine resistance mutations Met184Val/Ile of HIV-1 RT. In a departure from the pattern observed with HIV, 3TC-resistant HBV frequently contains a second polymerase mutation, Leu528Met. Met552Ile/Val mutations alone and in combination with the Leu528Met mutation confer a high degree of resistance to 3TC triphosphate (3TCTP) in vitro (Table ). On the other hand, ADV has been reported to be active against 3TC-resistant HBV in vitro and in vivo (29
). These data indicate complementary drug resistance profiles for 3TC and ADV against HBV, suggesting a potential advantage for combination therapy in treating chronic HBV infection where the emergence of resistance to either agent may be suppressed.
Inhibition of HBV polymerases containing prototypic 3TC resistance mutations
Knowledge of the structure of HBV polymerase would be valuable for understanding the molecular basis of many of its properties, including mechanisms of polymerization, inhibition, and drug resistance, and for interpretation of clinical and biochemical data. Attempts to determine the structure of HBV polymerase by various research groups have not yet been successful, as they have been limited by failure to obtain sufficient amounts of highly purified active protein.
The work presented here includes a molecular modeling study of HBV polymerase based on available retroviral RT structures. The validity of the model developed in the present study is supported by its ability to explain some of the key biochemical data. The inhibition potencies of 3TCTP, FTCTP, ADV diphosphate (ADVDP), PCVTP, and LBVTP were evaluated and compared with the Km for dCTP in in vitro enzyme assays for wild type HBV polymerase and a Leu528Met mutant, Met552Ile/Val mutants, and Leu528Met+Met552Ile/Val mutants. The results were analyzed at the atomic level using the modeled three-dimensional structure of HBV polymerase. dCTP, 3TCTP, FTCTP, and ADVDP were docked into the modeled enzyme so that the differential effects of Met552Ile/Val and Leu528Met mutations on different nucleotide analogs could be examined. Possible effects of these mutations on some other potent nucleotide inhibitors are addressed. Understanding of the roles of these drug resistance mutations might be helpful in achieving the broader goal of developing more effective antiviral strategies for the treatment of chronic hepatitis B.