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Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA
Yuanjie Liu,
Hui Nie,
Richeng Mao,
Bidisha Mitra,
Dawei Cai,
Ran Yan,
Ju-Tao Guo,
Timothy M. Block,
Nadir Mechti,
Haitao Guo
PLOS
Published: April 11, 2017
https://doi.org/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.
Author summary
HBV is a DNA virus but replicates its DNA via retrotranscription of a viral RNA pregenome. ISG20, an antiviral RNase induced by interferons, inhibits the replication of many RNA viruses but the underlying molecular antiviral mechanism remains elusive. Since all the known viruses, except for prions, have RNA products in their life cycles, ISG20 can be a broad spectrum antiviral protein; but in order to distinguish viral RNA from host RNA, ISG20 may have evolved to recognize virus-specific signals as its antiviral target. We demonstrated herein 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. Because ε is the HBV pregenomic RNA packaging signal and reverse transcription priming site, the binding of ISG20 to ε, even in the absence of ribonuclease activity, results in antiviral effect to prevent DNA replication due to preventing viral polymerase binding to pgRNA. We also 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.
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发表于 2017-4-18 18:17