title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.
abstract: Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Yuanjie
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Hui
authors.identifier: http://orcid.org/0000-0001-8827-8872
authors.initials: H
authors.lastname: Nie
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Richeng
authors.initials: R
authors.lastname: Mao
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Bidisha
authors.initials: B
authors.lastname: Mitra
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Dawei
authors.initials: D
authors.lastname: Cai
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Ran
authors.initials: R
authors.lastname: Yan
authors.affiliation: Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America.
authors.forename: Ju-Tao
authors.initials: JT
authors.lastname: Guo
authors.affiliation: Baruch S. Blumberg Institute, Doylestown, Pennsylvania, United States of America.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Block
authors.affiliation: CNRS, UMR5235, DIMNP, University of Montpellier 2, Montpellier, France.
authors.forename: Nadir
authors.initials: N
authors.lastname: Mechti
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
authors.forename: Haitao
authors.identifier: http://orcid.org/0000-0002-7146-916X
authors.initials: H
authors.lastname: Guo
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology; D005092:Exonucleases / Q000378:metabolism* / Q000494:pharmacology*; D005095:Exoribonucleases; D006515:Hepatitis B virus / Q000302:isolation & purification / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000821:virology; D006801:Humans; D020871:RNA Stability / Q000187:drug effects; D012367:RNA, Viral / Q000187:drug effects* / Q000378:metabolism; D048348:Reverse Transcription / Q000187:drug effects; D012260:Ribonucleases / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral; D005092:Exonucleases; D005095:Exoribonucleases; C112346:ISG20 protein, human; D012260:Ribonucleases