An antiviral drug-resistant mutant of hepatitis B virus with high replication capacity in association with a large in-fr
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ORIGINAL PAPER
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 Ting Wang1,5 · Yanli Qin2 · Jing Zhang3 · Xinyan Li4 · Shuping Tong3 · Weifeng Zhao5 · Jiming Zhang1,2 Received: 24 November 2019 / Accepted: 8 August 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords Hepatitis B virus · Hepatitis B surface antigen · Surface gene · PreS1 deletion · rtN236/rtA181 mutations · Replication capacity Abbreviations ADV Adefovir CHB Chronic hepatitis B CMV Cytomegalovirus DMEM Dulbecco’s modified Eagle medium Edited by Wolfram H. Gerlich. Ting Wang and Yanli Qin have contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11262-020-01787-9) contains supplementary material, which is available to authorized users. * Weifeng Zhao [email protected] * Jiming Zhang [email protected] Extended author information available on the last page of the article
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 Lamivudi
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