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Hepatology
Original research
RNA helicase DDX5 enables STAT1 mRNA translation and interferon signalling in hepatitis B virus replicating hepatocytes
  1. Jiazeng Sun1,
  2. Guanhui Wu2,
  3. Florentin Pastor3,
  4. Naimur Rahman4,
  5. Wen-Hung Wang5,
  6. Zhengtao Zhang6,
  7. Philippe MERLE7,
  8. Lijian Hui6,
  9. Anna Salvetti3,
  10. David Durantel8,
  11. Danzhou Yang2,
  12. Ourania Andrisani1
  1. 1 Basic Medical Sciences, Purdue University, West Lafayette, Indiana, USA
  2. 2 Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA
  3. 3 International Center for Infectiology Research (CIRI), INSERM U1111-CNRS UMR5308, Lyon, France
  4. 4 Basic Medical Sciences, Purdue University System, West Lafayette, Indiana, USA
  5. 5 Gene Editing Core, Bindley Biosciences Center, Purdue University, West Lafayette, Indiana, USA
  6. 6 Department of Biochemistry and Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China
  7. 7 Service d'Hépatologie, Hôpital de La Croix-Rousse Centre Livet, Lyon, Rhône-Alpes, France
  8. 8 INSERM U1111-CNRS UMR5308 International Center for Infectiology Research (CIRI), Lyon, France
  1. Correspondence to Dr Ourania Andrisani, Basic Medical Sciences, Purdue University, West Lafayette IN 47907, USA; andrisao{at}purdue.edu

Abstract

Objective RNA helicase DDX5 is downregulated during HBV replication and poor prognosis HBV-related hepatocellular carcinoma (HCC). The objective of this study is to investigate the role of DDX5 in interferon (IFN) signalling. We provide evidence of a novel mechanism involving DDX5 that enables translation of transcription factor STAT1 mediating the IFN response.

Design and results Molecular, pharmacological and biophysical assays were used together with cellular models of HBV replication, HCC cell lines and liver tumours. We demonstrate that DDX5 regulates STAT1 mRNA translation by resolving a G-quadruplex (rG4) RNA structure, proximal to the 5′ end of STAT1 5′UTR. We employed luciferase reporter assays comparing wild type (WT) versus mutant rG4 sequence, rG4-stabilising compounds, CRISPR/Cas9 editing of the STAT1-rG4 sequence and circular dichroism determination of the rG4 structure. STAT1-rG4 edited cell lines were resistant to the effect of rG4-stabilising compounds in response to IFN-α, while HCC cell lines expressing low DDX5 exhibited reduced IFN response. Ribonucleoprotein and electrophoretic mobility assays demonstrated direct and selective binding of RNA helicase-active DDX5 to the WT STAT1-rG4 sequence. Immunohistochemistry of normal liver and liver tumours demonstrated that absence of DDX5 corresponded to absence of STAT1. Significantly, knockdown of DDX5 in HBV infected HepaRG cells reduced the anti-viral effect of IFN-α.

Conclusion RNA helicase DDX5 resolves a G-quadruplex structure in 5′UTR of STAT1 mRNA, enabling STAT1 translation. We propose that DDX5 is a key regulator of the dynamic range of IFN response during innate immunity and adjuvant IFN-α therapy.

  • hepatitis B
  • hepatocyte
  • interferon-alpha
  • molecular mechanisms
  • hepatocellular carcinoma

Data availability statement

All data relevant to the study are included in the article or uploaded as supplementary information. All data are available for review.

https://doi.org/10.1136/gutjnl-2020-323126

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    Data availability statement

    All data relevant to the study are included in the article or uploaded as supplementary information. All data are available for review.

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    Footnotes

    • GW and FP contributed equally.

    • Contributors This work was primarily performed by JS. GY and and DY contributed the biophysical studies on experiments regarding rG4 structure. W-HW contributed the gene editing. ZZ and LH contributed the analyses of the HBV-HCC cell lines. FP, AS and DD contributed the HepaRG/HBV infection studies. PM contributed the clinically annotated HBV-HCC tissues. NR contributed the inducible DDX5-FLAG expressing cell lines. OA directed the studies and wrote the manuscript.

    • Funding This work was supported by NIH grants DK044533 to OA and CA177585 to DY. Shared Resources (Genomics and Bioinformatics Facilities) supported by NIH grant P30CA023168 to Purdue Center for Cancer Research, and NIH/NCRR RR025761.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.