RNA helicase DDX5 enables STAT1 mRNA translation and interferon signaling in hepatitis B virus replicating hepatocytes

Abstract

ObjectiveRNA 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 understand the significance of DDX5 downregulation during HBV infection and poor prognosis HCC, as it pertains to therapy of chronically HBV-infected patients with HCC. IFN- therapy is effective only for a subgroup of HBV-infected patients, for reasons not understood. Herein, we investigated a novel mechanism of STAT1 translational control, involving DDX5-mediated resolution of a G-quadruplex structure, located at the 5UTR of STAT1 mRNA, enabling STAT1 translation. Design and ResultsMolecular, pharmacologic, and biophysical assays were used together with cellular model of HBV replication, HCC cell lines, and HBV-related liver tumors. We observed, the protein level of DDX5 correlated with that of transcription factor STAT1. We show, DDX5 regulates STAT1 mRNA translation by resolving a G-quadruplex (rG4) RNA structure, proximal to the 5 end of STAT1 5UTR. We employed luciferase reporter assays comparing wild type (WT) vs. mutant (MT) rG4 sequence, rG4-stabilizing compounds, CRISPR/Cas9 editing of the rG4 sequence, and circular dichroism determination of rG4 structure. Ribonucleoprotein assays identified direct DDX5 binding to WT but not MT rG4. Importantly, liver cancer cell lines expressing low DDX5 exhibited reduced interferon response, while immunohistochemistry of HBV-related HCCs exhibiting absence of DDX5, also lacked STAT1. ConclusionDDX5 resolves a G-quadruplex structure in 5UTR of STAT1 mRNA, enabling STAT1 translation. DDX5 is a key regulator of the dynamic range of interferon response during innate immunity and adjuvant IFN- therapy.