Entecavir (ETV) exhibits potent antiviral activity in patients chronically infected with wild-type or lamivudine (3TC)-resistant (3TC r ) hepatitis B virus (HBV). Among the patients treated in phase II ETV clinical trials, two patients for whom previous therapies had failed exhibited virologic breakthrough while on ETV.Isolates from these patients (arbitrarily designated patients A and B) were analyzed genotypically for emergent substitutions in HBV reverse transcriptase (RT) and phenotypically for reduced susceptibility in cultures and in HBV polymerase assays. After 54 weeks of 3TC therapy, patient A (AI463901-A) received 0.5 mg of ETV for 52 weeks followed by a combination of ETV and 100 mg of 3TC for 89 weeks. Viral rebound occurred at 133 weeks after ETV was started. The 3TC r RT substitutions rtV173L, rtL180M, and rtM204V were present at study entry, and the additional substitutions rtI169T and rtM250V emerged during ETV-3TC combination treatment. Reduced ETV susceptibility in vitro required the rtM250V substitution in addition to the 3TC r substitutions. For liver transplant patient B (AI463015-B), previous famciclovir, ganciclovir, foscarnet, and 3TC therapies had failed, and RT changes rtS78S/T, rtV173L, rtL180M, rtT184S, and rtM204V were present at study entry. Viral rebound occurred after 76 weeks of therapy with ETV at 1.0 mg, with the emergence of rtT184G, rtI169T, and rtS202I substitutions within the preexisting 3TC r background. Reduced susceptibility in vitro was highest when both the rtT184G and the rtS202I changes were combined with the 3TC r substitutions. In summary, infrequent ETV resistance can emerge during prolonged therapy, with selection of additional RT substitutions within a 3TC r HBV background, leading to reduced ETV susceptibility and treatment failure.Nearly 400 million people are chronically infected with hepatitis B virus (HBV) worldwide (16,19). After prolonged infections, often lasting decades, patients frequently develop severe liver disease that can lead to cirrhosis and hepatocellular carcinoma. Chronically infected patients also serve as sources of HBV transmission. There are currently three approved therapies for chronic HBV infections: interferon, lamivudine (3TC; ␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine), and adefovir-dipivoxil, the prodrug of adefovir [ADV; 9-(2-phosphonylmethoxyethyl)adenine] (18). Interferon is administered subcutaneously and is associated with numerous adverse events, some of which can be severe, and a sustained antiviral response in only 30 to 40% of treated patients (30). 3TC treatment, administered orally, is effective in reducing viral loads but results in the frequent emergence of drug-resistant HBV due to substitutions at the Tyr-Met-Asp-Asp (YMDD) nucleotide binding site motif of viral DNA polymerase. Data from four large clinical trials revealed 3TC resistance (3TC r ) mutations in 24, 42, 53, and 70% of patients after 1, 2, 3, and 4 years of therapy, respectively (15). Treatment with ADV, recently approved by the U.S. Food and Drug Administration,...

The hepatitis B virus (HBV) mutation that encodes rtA181T is selected in the viral polymerase during antiviral drug therapy and can also encode a stop codon in the overlapping surface gene at amino acid 172 (sW172*) resulting in truncation of the last 55 amino acids of the C-terminal hydrophobic region of the surface proteins. This mutation is usually detected as a mixed population with wild-type HBV. In vitro analysis revealed that the rtA181T/ sW172* variant is not only defective in secretion of viral particles causing intracellular retention of surface proteins, it also has a dominant negative effect on virion but not subviral particle secretion when coexpressed with the wild type. This dominant negative effect was attributed to the truncated S protein alone. Furthermore, these truncated surface proteins were less glycosylated, and the truncated L protein was able to support virion secretion. Examination of sequential HBV DNA levels in patients failing lamivudine or adefovir therapy where only the rtA181T change was detected via polymerase chain reaction sequencing revealed that viral load rebound did not occur or was not as large as usually observed with drug-resistant HBV. Conclusion: The rtA181T/sW172* variant has a secretory defect and exerts a dominant negative effect on wild-type HBV virion secretion. The selection of rtA181T/sW172* reduced the typical extent of virological breakthrough, resulting in a missed diagnosis of drug resistance if viral load was used as the only criterion for drug failure, necessitating HBV polymerase chain reaction sequencing or other genotypic methods to diagnose antiviral drug resistance in these cases. (HEPATOLOGY 2008;48:88-98.) T reatments for hepatitis B virus (HBV) infection include interferon and nucleos(t)ide analogs (NAs). However, treatment with NAs is hampered by selection of changes in the HBV reverse transcriptase (rt) associated with resistance. The region of the HBV genome that encodes the rt domains of the polymerase overlaps completely with the envelope; therefore, changes associated with NA therapy can cause the selection of envelope changes. 1 HBV encodes three membrane-associated envelope proteins, all sharing the same stop codon, with translation initiating from different start codons. The smallest of these proteins, S, is encoded by the S gene, which produces a protein of 226 amino acids (aa). The middle protein, M, contains a further N-terminal extension of 55 aa encoded by the upstream Pre-S2 gene. The L protein has yet another N-terminal extension of 108-119 aa, depending on the genotype, encoded by Pre-S1. The major functions of the HBV surface proteins include envelopment of nucleocapsids with subsequent assembly of virions, and assembly into empty subviral particles that are secreted in great excess over HBV virions collectively referred to as hepatitis B surface antigen. The surface proteins also contain the major antigenic epitopes and are involved in hepatocyte binding and entry during infection.The nucleotide mutation that encodes rtA181T has ...

Long-term lamivudine (LMV) treatment of chronic hepatitis B almost inevitably engenders viral resistance.Mutations that result in the replacement of the methionine at position 204 of the deoxynucleoside triphosphate-binding site of the hepatitis B virus (HBV) reverse transcriptase (rt) by isoleucine, valine, or (rarely) serine (rtM204I/V/S) confer high-level resistance to LMV but reduce replication efficiency. The subsequent selection or coselection of secondary mutations that partially restore replication efficiency is common and may influence drug resistance. Genotyping has shown that LMV treatment can select for HBV rtL80V/I mutants, but their prevalence and phenotype have not been documented. Analysis of a large sequence database revealed that rtL80V/I occurred almost exclusively in association with LMV resistance, and 85% of these isolates encoded rtL80I. Coselection of rtL80V/I occurred in 46% of isolates in which LMV resistance was attributable to rtM204I but only 9% of those in which resistance was attributable to rtM204V. Moreover, rtL80V/I did not occur in HBV genotype A isolates but occurred at similar frequencies in genotype B, C, and D isolates. In vitro phenotyping showed that although the rtL80I mutant by itself replicated less efficiently and was hypersensitive to LMV compared to the replication efficiency and sensitivity of its wild-type parent, the presence of rtL80I enhanced the replication efficiency of rt204I/V mutants without significantly affecting LMV resistance. Molecular modeling revealed that rt80 does not interact directly with the enzyme's substrates. Collectively, these results suggest that coselection of rtL80V/I and rtM204I/V occurs because the former compensates for the loss of replication efficiency associated with the acquisition of LMV resistance, particularly in the case of rtM204I.The introduction in 1998 of lamivudine (LMV), the first safe and efficacious orally available inhibitor of hepatitis B virus (HBV) replication, revolutionized the treatment of chronic hepatitis B (CHB). LMV is a synthetic deoxycytidine analogue that is phosphorylated intracellularly by host cell enzymes which generate its 5Ј-triphosphate (LMV-TP). LMV-TP inhibits replication by competing with dCTP for incorporation into nascent viral DNA. Since LMV lacks a 3Ј-hydroxyl homologue, which is required for chain extension, incorporation of an LMV monophosphate residue causes immediate replication arrest. Treatment with LMV rapidly and significantly decreases viremia in a majority of patients with CHB and arrests or reverses liver disease in many, as indicated by the normalization of the results of liver function tests and of liver histology (18). Unfortunately, the long-term effectiveness of LMV is reduced by the development of viral resistance (1), which increases cumulatively at an annual rate of approximately 14 to 20% and occurs most frequently in individuals who are coinfected with human immunodeficiency virus (HIV) type 1 (20). Provided that the viral load is less than 6 log 10 copies/ml, most case...

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.hepatitis B virus | cellular inhibitor of apoptosis proteins | cIAP1 | cIAP2 | TNF I t is estimated that 2 billion people currently living in the world have been infected with hepatitis B virus (HBV), and among these, 360 million people are chronic carriers (1). HBV causes 780,000 deaths each year and is responsible for 50% and 33% of deaths attributable to liver cancer and cirrhosis, respectively (2). The host factors and molecular pathways that impact on HBV disease and clinical outcomes are not well-understood (3). What is becoming clear is that immunosuppressive agents and particularly, biological agents, including anti-TNF therapy, can cause major flares in HBV-related disease, leading to morbidity and mortality (4, 5). Animal models and particularly, immunocompetent mouse models of persistent HBV infection have been used to dissect host-pathogen interactions that influence infection outcomes (6-8). These animal models can be used to define host cell signaling and cell death pathways that contribute to the persistence or control of HBV infection.We induced HBV infection in two mouse models to examine the relevance of host factors in controlling infection. In a model that mimics partial control of infection, we were able to determine the importance of host cell signaling pathways through the use of gene-targeted mice. By identifying the relevant host cell signaling molecules that impact on HBV clinical outcomes, it may be possible to develop therapeutics that target host cell pathways and alter the course of HBV-related disease. ResultsChronic HBV Infection Can Be Mimicked in a Mouse Model. We used a previously described method to induce HBV persistence in immunocompetent mice (6). A plasmid containing a 1.2 over length sequence of HBV genotype A was hydrodynamically injected into mice, but in contrast to the previously published protocol, we did not anesthetize animals. Using this modified technique, we did not observe any injection-associated mortality, and C57BL/6 mice showed persistently high serum HBV DNA levels over 8-12 wk (Fig. 1A). Eventually, HBV DNA levels fell in all animals along with t...