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Hepatitis B virus X gene mutants emerge during antiviral therapy and increase cccDNA levels to compensate for replication suppression

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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).

Author information

Authors and Affiliations

  1. Liver Research Unit, Keelung Chang Gung Memorial Hospital, Keelung, Taiwan

    Chih-Lang Lin & Rong-Nan Chien

  2. Liver Research Center, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan

    Chih-Lang Lin, Rong-Nan Chien, Yu-De Chu, Ya-Hui Huang, Yang-Hsiang Lin & Chau-Ting Yeh

  3. College of Medicine, Chang Gung University, Taoyuan, Taiwan

    Chih-Lang Lin, Rong-Nan Chien, Ya-Hui Huang, Yang-Hsiang Lin & Chau-Ting Yeh

  4. Community Medicine Research Center, Keelung Chang Gung Memorial Hospital, Keelung, Taiwan

    Chih-Lang Lin & Rong-Nan Chien

  5. Medical Research Department, Taipei Veterans General Hospital, Taipei, Taiwan

    Kung-Hao Liang & Chau-Ting Yeh

  6. Department of Biochemistry, Chang Gung University, Taoyuan, Taiwan

    Po-Yuan Ke, Kwang-Huei Lin & Chau-Ting Yeh

Authors
  1. Chih-Lang Lin
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  2. Rong-Nan Chien
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  9. Chau-Ting Yeh
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Contributions

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.

Corresponding authors

Correspondence to Yang-Hsiang Lin or Chau-Ting Yeh.

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Conflict of interest

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.

Informed consent

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

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