Abstract
Background
Hepatitis B virus (HBV) X gene (HBx) mutants can develop during the natural course of chronic HBV infection. However, little is known about whether the emergence of HBx mutants during long-term antiviral therapy is an adaptation of HBV to antiviral stress. This study was to identify HBx mutants that emerged in patients experiencing Lamivudine resistance or suboptimal treatment.
Methods
Forty-six Lamivudine-resistant patients and 46 patients with suboptimal treatment responses to Entecavir were enrolled in this study. HBx mutants were identified by sequence analysis and their roles in the HBV replication cycle were characterized.
Results
We show that deletion/truncation/insertion mutations were only detected in the Lamivudine resistance group, while synonymous mutations were found in both groups. Follow-up analyses revealed that five patients in the Lamivudine group developed hepatocellular carcinoma, while patients in the Entecavir group did not. These mutants were characterized by a significant decrease in transactivation of the pre-S1 promoter, and varying effects on transactivation of the X promoter. Co-transfection of HBx-mutant plasmid and HBV replication-competent clone into HepG2 cells resulted in increased nuclear-to-cytoplamic HBV core antigen, HBV-DNA ratios, and nuclear covalently closed circular DNA (cccDNA). Antiviral drug sensitivity assays revealed that these mutants exhibited a compensatory effect to counteract antiviral drug suppression, resulting in elevated secretory HBV-DNA levels.
Conclusions
Our study demonstrates that HBx mutants can emerge during Lamivudine or Entecavir therapy. These mutants exhibit altered transactivation of the HBV pre-S1 and X promoters, leading to increased cccDNA levels to compensate for replication suppression.
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Abbreviations
- HBx:
-
Hepatitis B virus (HBV) X gene
- cccDNA:
-
Nuclear covalently closed circular DNA
- HBV:
-
Hepatitis B virus
- HCC:
-
Hepatocellular carcinoma
- LAM:
-
Lamivudine
- ETV:
-
Entecavir
- Ct-HBx:
-
C-terminally truncated HBx
- HBsAg:
-
Hepatitis B surface antigen
- PC:
-
Core promoter (PC)
- PS1:
-
Pre-S1 promoter
- PS2:
-
Pre-S2/S promoter
- PX:
-
X promoter
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Acknowledgements
The authors appreciate the technical and administrative support provided by all members of the Liver Research Center in Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Funding
This study was supported by grants from Chang Gung Memorial Hospital, Taiwan (CMRPG2B0463 and CRRPG2H0081).
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CTY designed and supervised the study; CLL, RNC and YDC drafted the manuscript; YDC, YHH, KHL and PYK performed the experiments; CTY, CLL, KHL, YHL interpreted the data; CTY, CLL, RNC and YDC collected and analyzed the clinical data.
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No potential conflicts of interest were reported by the authors (Chih-Lang Lin, Rong-Nan Chien, Yu-De Chu, Kung-Hao Liang, Ya-Hui Huang, Po-Yuan Ke, Kwang-Huei Lin, Yang-Hsiang Lin, Chau-Ting Yeh).
Ethics approval statement
This study was approved by the Institutional Review Board of Chang Gung Medical Center. The experiments conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
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Informed consent was obtained from patients, prior to their participation in the study.
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YHL and CTY are co-corresponding authors.
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Lin, CL., Chien, RN., Chu, YD. et al. Hepatitis B virus X gene mutants emerge during antiviral therapy and increase cccDNA levels to compensate for replication suppression. Hepatol Int 14, 973–984 (2020). https://doi.org/10.1007/s12072-020-10079-1
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DOI: https://doi.org/10.1007/s12072-020-10079-1