The Inhibitory Effect Of Innate Immune Sensor IFI16 On HBV CccDNA And Its Underlying Mechanisms

Hepatitis B virus(HBV)infection remains still a major health threat worldwide,which may cause a wide range of liver diseases,including acute/chronic hepatitis,liver cirrhosis,and even hepatocellular carcinoma.HBV is a small(3.2-kb)DNA virus,which comprises a relaxed circular partially double-stranded DNA(rcDNA)genome.After entrying into hepatocytes,the rcDNA is delivered into the nucleus and converted into covalently closed circular DNA(cccDNA)which forms a minichromosome by histone and nonhistone proteins.HBV cccDNA serves as a template for the synthesis of all HBV RNA transcripts,including subgenomic RNAs and the pregenomic RNA(pgRNA),that functions as the template for reverse transcription into newly generated rcDNA.The rcDNA can be enveloped through the endoplasmic reticulum and Golgi complex followed by secretion from the cells or retransported into the nucleus for the abundance of the cccDNA pool.It is now evident that transcription of the pgRNA from the cccDNA is the pivotal step for HBV genome amplification and eventually determines the rate of HBV replication.It is now well-established that the therapeutic efficacy of current antiviral agents for HBV,including nucleoside analogues and IFN-a,is limited due to the persistence of viral cccDNA.Thus,the development of new therapeutic strategy for controlling cccDNA in hepatocytes is one of the major goals of HBV research.Accumulating evidence indicates that regulation of host immune response may play an important role in controlling HBV infection,including cccDNA inhibition.And pattern recognition receptors(PRRs),which recognize various pathogen-associated molecule patterns,have been shown to play a critical role in the initiation of antiviral innate immune response against pathogens.However,it remains still unclear whether and how HBV cccDNA is sensed by the innate immune sensors and whether the recognition of cccDNA by PRR contributes to the host defense against HBV.In this study,we investigated the potential PRR for the nuclear HBV cccDNA by ChIP analysis,Our data showed that IFI16,but not other nucleic acid sensors including cGAS,AIM2 and MNDA,was recruited to cccDNA microchromosome significantly in hepatocytes.Our results also showed that HBV markedly downregulate IFI16 mRNA and protein level,indicating there might exist interplay between HBV and IFI16.Overexpression of IFI 16 was found to inhibit cccDNA-driven HBV transcription and replication,as revealed by the downregulation of HBV proteins(HBsAg and HBeAg),HBV transcripts(pgRNA and HBV RNAs)and HBV DNA levels in IFI16-transfected cells,and we got similar results in HBV cccDNA mouse model treated with p204,the mouse homologue of IFI16.Further knockdown analysis showed that downregulation of IFI 16 significantly enhanced cccDNA-driven HBV transcription and replication.Mechanitically,it was found that,although being an AIM2-like receptor,IFI 16 showed little effect on inflammasome activation,while significantly activated IFN signaling in HBV cccDNA cell model,Further study revealed that although siRNA-mediated downregulation of IRF3 efficiently suppressed IFI16-activated IFN signaling,it didn't reverse IFI16-mediated inhibition of cccDNA significantly,indicating there might exist other mechanisms responsible for IFI 16 inhibition of HBV cccDNA function.Evidence indicates that HBV cccDNA-mediated transcription and replication are under control of epigenetic modification,while IFI 16 has been revealed to inhibit viral replication through epigenetic regulation,we thus investigated the effect of IFI 16 on the epigenetic modification of HBV cccDNA.Results showed IFI 16 treatment significantly decreased the acetylation of cccDNA-bound histone H3 and H4,which have been reported to be closely associated with HBV replication.Of note,we also observed that,upon IFI 16 expression,the recruitment of H3K4me3(a marker of euchromatin)to cccDNA was downregulated,while that of H3K9me3(a mark of heterochromatin)was upregulated significantly,indicating that IFI 16 treatment enhanced the heterochromatinization of cccDNA.Further mechanistic study showed that IFI16 expression significantly inhibited the binding of STAT1/STAT2 to the interferon-stimulated response element(ISRE).Once the ISRE was mutated,the effect of IFI 16 on the epigenetic modification of cccDNA was severely impaired as demonstrated by the attenuation of IFI16-mediated effect on the recruitment of STAT1,STAT2,AcH3,H3K4me3 and H3K9me3 to cccDNA.And as expected,ISRE mutation significantly decreased the inhibitory effect of IFI16 on cccDNA-driven HBV transcription and replication,indicating that HBV ISRE might mediate the transcripitional repression of IFI16.In summary,our results firstly demonstrated that innate nucleic acid sensor IFI 16 could recognize HBV cccDNA in the nucleus of hepatocytes,and repressed cccDNA-driven HBV transcription and replication.Upon binding to cccDNA,IFI16 had little effect on inflammasome activation,but activated IFN signaling pathway.Of note,IFI16 might act as a direct restriction factor for HBV though epigenetic regulation of cccDNA.Together,our data indicated that targeting IFI16 might provide a novel therapeutic strategy for controlling persistent HBV infection.