The Impact of the Hepatitis B Virus Polymerase rtA181T Mutation on Replication and Drug Resistance Is Potentially Affected by Overlapping Changes in Surface Gene

Abstract

The emergence of drug-resistant hepatitis B virus (HBV) is a major problem for antiviral treatment in chronic hepatitis B infection. In this study, we analyzed the evolution of drug-resistant mutations and characterized the effects of the rtA181T and rtI233V mutations on viral replication and drug resistance. We performed a clonal analysis of the HBV polymerase gene from serum samples during viral breakthrough treated with antiviral agents. A series of mutant clones containing rtA181T and/or rtI233V mutations were constructed and determined the effect of these mutations on the replication ability and drug resistance. An in vitro study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. Compared to the rtA181T surface missense mutation (rtA181T/sW172S), the introduction of rtA181T surface nonsense mutation (rtA181T/sW172*) resulted in decreased viral replication and increased drug resistance. Complementation assay revealed that the truncated PreS1 is responsible for reduced replication of rtA181T/sW172* mutant. Moreover, the rtA181T/sW172* mutant exhibited a defect in viral particle secretion. The rtI233V mutation that emerged during adefovir therapy reduced viral replication and conferred resistance to adefovir. Our data suggest that the impact of the rtA181T mutation on replication and drug resistance differs based on the mutation status of the corresponding surface gene. The rtI233V mutation also affects replication ability and drug resistance. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. IMPORTANCEThe emergence of drug-resistant HBV that are no longer susceptible to nucleos(t)ide analogues is a major problem for antiviral treatment in chronic hepatitis B infection. Among drug-resistant mutations, the single rtA181T mutation is known to confer cross-resistance to antiviral drugs. This mutation causes intermediate or reduced susceptibility to tenofovir. Moreover, the clinical occurrence of the rtA181T mutation during antiviral therapy is also high. Our study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. We believe that our study will not only extend the understanding of the drug resistance mechanism, but it will also ultimately provide new treatment options for patients with multidrug resistant HBV.

Resistance mutations of hepatitis B virus in entecavir‐refractory patients

“…This mutation has been reported to enhance the replication efficiency and to be associated with enhanced replication efficiency when combined with the rtM204I mutation, although rtL269I alone did not alter susceptibilities to NAs 25, 27. In our experiment, rtL269I but not rtN238H could enhance ETV resistance when combined with rtL180Q/M204V, which is consistent with previous data 27, 28…”

“…rtA181T mutation‐associated ADV resistance has been reported 28, 32, 33, 34. This mutation is often associated with a stop codon at the overlapping surface gene, and the sW172stop mutation impairs HBsAg secretion by generating a truncated protein and also causes the susceptibility to ADV to deteriorate.…”

Possible Involvement of Multidrug-Resistant Hepatitis B Virus sW172* Truncation Variant in the ER Stress Signaling Pathway during Hepatocarcinogenesis

“…The selection of rtA181T/sW172 * multi-drug resistant mutants by widespread treatment with NAs has raised many concerns due to decreased HBV susceptibility to LMV, LdT, ADV, and tenofovir (8,10,20). Its dominant negative effect on wild-type HBV virion secretion and oncogenic potential through transactivation activities were also reported (10)(11)(12).…”

Biological characteristics comparison of HBV rtA181T mutants with truncated or substituted HBsAg expression in vitro and in vivo model systems

“…However, it is worth to mention that the viral replication changes of this mutant are inconsistent in different in vitro cell studies. For example, the result reported by Dr.Hiromi Yatsuji showed that there was no noticeable difference in replication ability between the rtA181T/sW172* and rtA181T/sW172L12; while other two in vitro studies reported a reduced replication of rtA181T/sW172* mutant1314. Thus, to better and fully understand the characteristics of the HBV rtA181T mutant, we designed this study to investigate the impact of different overlapping S gene mutation (sW172L and sW172*) on rtA181T mutant DNA replication and viral protein expression, not only in vitro in cultured HepG2 cells but also in vivo in mouse model.…”