RNA Helicase DDX5 in Association With IFI16 and the Polycomb Repressive Complex 2 Silences Transcription of the Hepatitis B Virus by Interferon

Abstract

RNA helicase DDX5 is a host restriction factor for hepatitis B virus (HBV) biosynthesis. Mass spectrometry (LC-MS/MS) identified significant DDX5-interacting partners, including interferon-inducible protein 16 (IFI16) and RBBP4/7, an auxiliary subunit of polycomb repressive complex 2 (PRC2). DDX5 co-eluted with IFI16, RBBP4/7, and core PRC2 subunits in size exclusion chromatography fractions derived from native nuclear extracts. Native gel electrophoresis of DDX5 immunoprecipitants revealed a 750 kDa DDX5/IFI16/PRC2 complex, validated by nanoscale co-localization via super-resolution microscopy. Prior studies demonstrated that IFI16 suppresses HBV transcription by binding to the interferon-sensitive response element of covalently closed circular DNA (cccDNA), reducing H3 acetylation and increasing H3K27me3 levels by an unknown mechanism. Herein, we demonstrate that ectopic expression of IFI16 inhibited HBV transcription from recombinant rcccDNA, correlating with increased IFI16 binding to rcccDNA, reduced H3 acetylation, and elevated H3K27me3, determined by chromatin immunoprecipitation. Importantly, the inhibitory effect of ectopic IFI16 on HBV transcription was reversed by siRNA-mediated knockdown of DDX5 and EZH2, the methyltransferase subunit of PRC2. This reversal was associated with decreased IFI16 binding to rcccDNA, enhanced H3 acetylation, and reduced H3K27me3. Similarly, endogenous IFI16 induced by interferon-α inhibited HBV rcccDNA transcription in a DDX5- and PRC2-dependent manner. In HBV-infected HepG2-NTCP cells, the antiviral effect of interferon-α was abrogated upon knockdown of DDX5 and EZH2, underscoring the crucial role of the DDX5 complex in IFI16-mediated antiviral response. In conclusion, in response to interferon, DDX5 partners with IFI16 to bind cccDNA, directing PRC2 to epigenetically silence cccDNA chromatin, thereby regulating immune signaling and HBV transcription.

Keywords: RNA helicase DEAD box protein 5; interferon Inducible protein 16; polycomb repressive complex 2; pyrin and hematopoietic interferon‐inducible nuclear (HIN) domain (PYHIN) family; single‐molecule localization microscopy (SMLM).