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An antiviral drug-resistant mutant of hepatitis B virus with high replication capacity in association with a large in-frame deletion in the preS1 region of viral surface gene

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Abstract

We amplified a full-length hepatitis B virus (HBV) genome from the serum of a chronic hepatitis B patient who experienced virological breakthrough with high HBV DNA titer following adefovir (ADV) therapy. The PCR product was cloned and sequencing of the six clones revealed an isolate of C2 subgenotype. Mutation(s) in the polymerase gene responsible for ADV resistance included rtA181T (all clones) and rtN236T (four clones). The rtA181T mutation caused the W172* nonsense mutation in the overlapping S gene. In addition, all the clones harbored another nonsense mutation in the S gene (C69*) and a 207nt in-frame deletion in the preS1 region. These clones were converted to a 1.1mer construct for transient transfection of Huh7 cells. All the clones were deficient in hepatitis B surface antigen production. Three clones had similar levels of DNA replication. Comparison with a wild-type clone of the same genotype revealed a higher intracellular level of replicative DNA for clone c4, which was reduced by putting back the deleted 207nt, but not by co-transfection with an expression construct for the three surface proteins to rescue virion production. The HBcAg expression of the c4 and c4+207nt clones was mainly in the nucleus. Co-transfection with the L/M/S proteins expression construct did not alter the distribution of core. Clone c4 showed a significantly decreased susceptibility to ADV, a mild reduction in susceptibility to lamivudine and tenofovir, but remained sensitive to entecavir. In conclusion, this is an unusual ADV-resistant HBV isolate harboring two nonsense mutations in the S gene and a large in-frame deletion in the preS1 region, but still retains a high replication phenotype, which can provide a platform for recombinant vector construction.

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Abbreviations

ADV:

Adefovir

CHB:

Chronic hepatitis B

CMV:

Cytomegalovirus

DMEM:

Dulbecco’s modified Eagle medium

ELISA:

Enzyme-linked immunosorbent assay

ETV:

Entecavir

HBeAg:

Hepatitis B e antigen

HBsAg:

Hepatitis B surface antigen

HBV:

Hepatitis B virus

HCC:

Hepatocellular carcinoma

LAM:

Lamivudine

L, M, S:

Large, middle, and small surface proteins

ORF:

Open reading frame

P:

Polymerase

PCR:

Polymerase chain reaction

pgRNA:

Pregenomic RNA

RT:

Reverse transcriptase

TDF:

Tenofovir

WT:

Wild type

WTB:

Wild-type clone of subgenotype B2

WTC:

Wild-type clone of subgenotype C2

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Acknowledgements

This work was supported by Shanghai Key Clinical Specialty Construction Program (ZK2019B24); National Natural Science Foundation of China (81101241, 81672009, 81871640); and Shanghai Pujiang Talent Plan12PJ1401600.

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Author notes

  1. Ting Wang and Yanli Qin have contributed equally to this work.

Authors and Affiliations

  1. Department of Infectious Diseases, Jing’An District Centre Hospital of Shanghai (Huashan Hospital, Fudan University Jing’An Branch), Shanghai, China

    Ting Wang & Jiming Zhang

  2. Department of Infectious Diseases, Huashan Hospital, Fudan University, 12 Middle Wulumuqi Road, Shanghai, 200040, China

    Yanli Qin & Jiming Zhang

  3. Key Lab of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China

    Jing Zhang & Shuping Tong

  4. Department of Hepatitis Diseases, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China

    Xinyan Li

  5. Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, 188 Shizi Street, Gusu District, Soochow, 215006, Jiangsu, China

    Ting Wang & Weifeng Zhao

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  1. Ting Wang
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  2. Yanli Qin
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Contributions

Jiming Zhang and Weifeng Zhao designed experiments. Ting Wang, Yanli Qin, Jing Zhang, and Xinyan Li performed experiments and analysis. Ting Wang, Yanli Qin, Shuping Tong, and Jiming Zhang wrote the manuscript. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Weifeng Zhao or Jiming Zhang.

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Wang, T., Qin, Y., Zhang, J. et al. An antiviral drug-resistant mutant of hepatitis B virus with high replication capacity in association with a large in-frame deletion in the preS1 region of viral surface gene. Virus Genes 56, 677–686 (2020). https://doi.org/10.1007/s11262-020-01787-9

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