Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA.

Yuanjie Liu,Timothy M Block,Hui Nie,Bidisha Mitra,Haitao Guo,Nadir Mechti,Richeng Mao,Ju-Tao Guo,Ran Yan,Dawei Cai

doi:10.1371/journal.ppat.1006296

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.

Highlights

Hepatitis B virus (HBV) infection remains a significant health threat to humans, leading to the elevated rate of severe liver diseases, such as fulminant hepatitis, fibrosis, cirrhosis, primary hepatocellular carcinoma, and other clinical complications [1].HBV is the prototype member of hepadnaviridae family which contains a number of DNA viruses replicating their genome through reverse transcription of a viral RNA intermediate in hepatocytes
We demonstrated that ISG20 selectively binds to a unique stem-loop structure called epsilon (ε) in all HBV RNA species and degrades viral RNA to inhibit HBV replication
We determined the structure and sequence requirements of ε RNA and ISG20 protein for ISG20-ε binding and antiviral activity. Such information will aid the function study of ISG20 against viral pathogens in host innate defense, and ISG20 has potentials to be developed into a therapeutic agent for viral diseases including hepatitis B

Summary

Introduction

HBV is the prototype member of hepadnaviridae family which contains a number of DNA viruses replicating their genome through reverse transcription of a viral RNA intermediate in hepatocytes. Besides serving as the template for translation of viral core protein and polymerase (pol), the pgRNA is the template for reverse transcription. The pol recognizes a stem loop structure (epsilon, ε) at the 5’ terminus of pgRNA to recruit core proteins to encapsidate pol/pgRNA complex into nucleocapsid, where the reverse transcription takes place to yield progeny viral rcDNA [3]. Considering the importance of viral RNA in HBV life cycle, it is conceivable that HBV RNA reduction will result in a suppression of both viral DNA replication and antigen production. In terms of interferon-stimulated genes (ISG), the tripartite motif-containing protein 22 (TRIM22) and DEAD-box RNA helicase DDX3 have been shown to inhibit HBV RNA transcription [8, 9], and the zinc finger antiviral protein (ZAP) and myeloid differentiation primary response gene 88 (MyD88) have been identified as host intrinsic antiviral factors against HBV through promoting the decay of HBV RNA [10, 11]

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