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

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 virus entry into HepG2-NTCP cells requires clathrin-mediated endocytosis.

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP-overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2-NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae-mediated endocytosis. Instead, entry occurred via the clathrin-mediated endocytosis pathway. HBV internalisation was inhibited by pitstop-2 treatment and RNA-mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP-2 and dynamin-2. We were able to visualise HBV entry in clathrin-coated pits and vesicles by electron microscopy (EM) and cryo-EM with immunogold labelling. These data demonstrating that HBV uses a clathrin-mediated endocytosis pathway to enter HepG2-NTCP cells increase our understanding of the complete HBV life cycle.

Direct interaction between the hepatitis B virus core and envelope proteins analyzed in a cellular context.

Hepatitis B virus (HBV) production requires intricate interactions between the envelope and core proteins. Analyses of mutants of these proteins have made it possible to map regions involved in the formation and secretion of virions. Tests of binding between core and envelope peptides have also been performed in cell-free conditions, to study the interactions potentially underlying these mechanisms. We investigated the residues essential for core-envelope interaction in a cellular context in more detail, by transiently producing mutant or wild-type L, S, or core proteins separately or in combination, in Huh7 cells. The colocalization and interaction of these proteins were studied by confocal microscopy and co-immunoprecipitation, respectively. The L protein was shown to constitute a molecular platform for the recruitment of S and core proteins in a perinuclear environment. Several core amino acids were found to be essential for direct interaction with L, including residue Y132, known to be crucial for capsid formation, and residues L60, L95, K96 and I126. Our results confirm the key role of L in the tripartite core-S-L interaction and identify the residues involved in direct core-L interaction. This model may be valuable for studies of the potential of drugs to inhibit HBV core-envelope interaction.