RNA helicase DDX5 enables STAT1 mRNA translation and interferon signaling in hepatitis B virus replicating hepatocytes
Résumé
Objective: RNA helicase DDX5 is downregulated during hepatitis B virus (HBV) replication, and poor prognosis HBV-related hepatocellular carcinoma (HCC). The objective of this study is to investigate the role of DDX5 in interferon signaling. We provide evidence of a novel mechanism involving DDX5 that enables translation of transcription factor STAT1 mediating the interferon (IFN) response.
Design and Results: Molecular, pharmacologic, and biophysical assays were used together with cellular models of HBV replication, HCC cell lines, and liver tumors. 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) vs. mutant (MT) rG4 sequence, rG4-stabilizing 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-stabilizing compounds in response to IFN-α, while HCC cell lines expressing low DDX5 exhibited reduced interferon 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 tumors 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 interferon response during innate immunity and adjuvant IFN-α therapy.
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