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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

Effect of rtL269I and other substitutions on resistance to lamivudine (LMV).(A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P < 0.05). The relative replication levels of each HBV construct was shown as the mean value of at least three independent experiments.
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pone.0136728.g003: Effect of rtL269I and other substitutions on resistance to lamivudine (LMV).(A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P < 0.05). The relative replication levels of each HBV construct was shown as the mean value of at least three independent experiments.

Mentions: The effect of rtL269I substitution on drug resistance was then investigated. As the quintuple mutant was resistant to LMV and ETV (Fig 1C), the effect of rtL269I on LMV (Fig 3) and ETV (Fig 4) resistance was determined using all artificial mutants.


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)

Effect of rtL269I and other substitutions on resistance to lamivudine (LMV).(A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P < 0.05). The relative replication levels of each HBV construct was shown as the mean value of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556173&req=5

pone.0136728.g003: Effect of rtL269I and other substitutions on resistance to lamivudine (LMV).(A) HBV DNA constructs were transfected into Huh7 cells, which were treated with LMV for 3 days. The intracellular HBV DNA was prepared for Southern blot analysis. A representative result has been shown. (B) The relative replication levels of each HBV mutant (no drug vs LMV treatment) were calculated based on the results of Figs 2C and 3A. (C) The relative replication ability of the HBV mutants treated with LMV were determined by Southern blotting and quantified by Phosphorimager (*, P < 0.05). The relative replication levels of each HBV construct was shown as the mean value of at least three independent experiments.
Mentions: The effect of rtL269I substitution on drug resistance was then investigated. As the quintuple mutant was resistant to LMV and ETV (Fig 1C), the effect of rtL269I on LMV (Fig 3) and ETV (Fig 4) resistance was determined using all artificial mutants.

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