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Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

Ahn SH, Kim DH, Lee AR, Kim BK, Park YK, Park ES, Ahn SH, Shin GC, Park S, Kang HS, Rhee JK, Yang SI, Chong Y, Kim KH - PLoS ONE (2015)

Bottom Line: The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF).The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV.Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea.

ABSTRACT
The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

No MeSH data available.


Related in: MedlinePlus

rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV).(A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P < 0.01; ***, P < 0.001).
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pone.0136728.g002: rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV).(A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P < 0.01; ***, P < 0.001).

Mentions: A series of artificial mutants containing part of the quintuple mutation were constructed (Fig 2A). First, the effect of the rtM129L, rtV173L, and rtH337N mutations on the replication of the rtM204I YMDD mutant was tested. Southern blot analysis of four mutants (rtM204I, rtV173L+M204I, rtM129L+V173L+M204I, and rtM129L+V173L+M204I+H337N) was performed to determine the replicative capacity of each clone compared to the WT virus (Fig 2B). As expected, replication of the rtM204I mutant was barely detectable (less than 10% of the WT) [10]. Consistent with previous report [20], rtV173L facilitated replication of the rtM204I mutant. On the other hand, rtM129L and rtH337N did not increase replication of the YMDD mutant (Fig 2B).


Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

Ahn SH, Kim DH, Lee AR, Kim BK, Park YK, Park ES, Ahn SH, Shin GC, Park S, Kang HS, Rhee JK, Yang SI, Chong Y, Kim KH - PLoS ONE (2015)

rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV).(A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P < 0.01; ***, P < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556173&req=5

pone.0136728.g002: rtL269I substitution enhances the replication of both WT and drug-resistant hepatitis B virus (HBV).(A) Schematic diagram of each HBV mutant construct used in this study. (B–C) Effect of mutations at positions 204, 173, 129, 337, and 269 on HBV DNA replication. Huh7 cells cultured in six-well plates were transfected with HBV plasmids. HBV DNA levels were analyzed by Southern blotting. HBeAg in culture supernatant was determined by ELISA. (D) Phosphor-imager analysis of the relative replication capacities of the HBV mutants. The standard deviation from three independent experiments was calculated (**, P < 0.01; ***, P < 0.001).
Mentions: A series of artificial mutants containing part of the quintuple mutation were constructed (Fig 2A). First, the effect of the rtM129L, rtV173L, and rtH337N mutations on the replication of the rtM204I YMDD mutant was tested. Southern blot analysis of four mutants (rtM204I, rtV173L+M204I, rtM129L+V173L+M204I, and rtM129L+V173L+M204I+H337N) was performed to determine the replicative capacity of each clone compared to the WT virus (Fig 2B). As expected, replication of the rtM204I mutant was barely detectable (less than 10% of the WT) [10]. Consistent with previous report [20], rtV173L facilitated replication of the rtM204I mutant. On the other hand, rtM129L and rtH337N did not increase replication of the YMDD mutant (Fig 2B).

Bottom Line: The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF).The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV.Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Center for Cancer Research and Diagnostic Medicine, IBST, School of Medicine, Konkuk University, Seoul, Korea.

ABSTRACT
The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

No MeSH data available.


Related in: MedlinePlus