Viral DNA-Dependent Induction of Innate Immune Response to Hepatitis B Virus in Immortalized Mouse Hepatocytes

Abstract

Hepatitis B virus (HBV) infects hundreds of millions of people worldwide and causes acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV is an enveloped virus with a relaxed circular (RC) DNA genome. In the nuclei of infected human hepatocytes, conversion of RC DNA from the incoming virion or cytoplasmic mature nucleocapsid (NC) to the covalently closed circular (CCC) DNA, which serves as the template for producing all viral transcripts, is essential to establish and sustain viral replication. A prerequisite for CCC DNA formation is the uncoating (disassembly) of NCs to expose their RC DNA content for conversion to CCC DNA. We report here that in an immortalized mouse hepatocyte cell line, AML12HBV10, in which RC DNA exposure is enhanced, the exposed viral DNA could trigger an innate immune response that was able to modulate viral gene expression and replication. When viral gene expression and replication were low, the innate response initially stimulated these processes but subsequently acted to shut off viral gene expression and replication after they reached peak levels. Inhibition of viral DNA synthesis or cellular DNA sensing and innate immune signaling diminished the innate response. These results indicate that HBV DNA, when exposed in the host cell cytoplasm, can function to trigger an innate immune response that, in turn, modulates viral gene expression and replication. IMPORTANCE Chronic infection by hepatitis B virus (HBV) afflicts hundreds of millions worldwide and is sustained by the episomal covalently closed circular (CCC) DNA in the nuclei of infected hepatocytes.Release of viral genomic DNA from cytoplasmic nucleocapsids (NCs) (NC disassembly or uncoating) is a prerequisite for its conversion to CCC DNA, which can also potentially expose the viral DNA to host DNA sensors and trigger an innate immune response. We have found that in an immortalized mouse hepatocyte cell line in which efficient CCC DNA formation was associated with enhanced exposure of nucleocapsid-associated DNA, the exposed viral DNA indeed triggered host cytoplasmic DNA sensing and an innate immune response that was able to modulate HBV gene expression and replication. Thus, HBV can, under select conditions, be recognized by the host innate immune response through exposed viral DNA, which may be exploited therapeutically to clear viral persistence. Hepatitis B virus (HBV) has infected approximately 2 billion people worldwide, with 350 million of them becoming chronically infected (1). Annually, 1 million fatalities are attributed to acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) caused by HBV. HBV is a small, enveloped DNA virus that contains a 3.2-kb, partially double-stranded (DS), relaxed circular (RC) DNA genome and replicates via an RNA intermediate, the pregenomic RNA (pgRNA). Upon infection, HBV RC DNA is converted to covalently closed circular (CCC) DNA in the nuclei of infected human hepatocytes, which serves as the transcriptional template for all viral RNAs, incl...

Hepatitis B Virus and DNA Stimulation Trigger a Rapid Innate Immune Response through NF-κB

“…A recent study has proposed that HBV triggers antiviral innate immunity through retinoic acid-inducible gene (RIG-I) activation from RNA produced from HBV's DNA genome (30). In addition, other recent studies have demonstrated a role for the STING-TBK1-IRF3 in detection of HBV (35,36). Our studies were unable to substantiate a role for the STING pathway in early HBV responses.…”

“…To further investigate the recognition of foreign DNA in hepatocytes, we also examined the stimulator of IFN genes (STING) pathway that has been shown to be important for DNA responses and innate responses to HBV (17,35,36). Interestingly, knockdown of STING and the associated molecule cGAS and downstream proteins TBK1 and IRF3 had dramatic effects on gene induction by ISD, demonstrating almost complete inhibition (Fig.…”

MITA/STING and Its Alternative Splicing Isoform MRP Restrict Hepatitis B Virus Replication

“…Consistently, Cui et al . showed that HBV DNA could be detected by DNA sensors, specifically the cGAS/STING signaling pathway, in an immortalized mouse hepatocyte cell line [19]. On the other hand, HBV was shown to counteract the MITA/STING-mediated antiviral responses by disrupting K63-linked ubiquitination of MITA/STING [24].…”

“…However, it is possible that HBV is detected by the innate immune system, while the virus can actively suppress or evade the early antiviral response. Indeed, recent studies have shown that the pgRNA (pregenomic RNA) of HBV and the cytoplasmic exposed HBV rcDNA (relaxed circular DNA), a result of nucleocapsid destabilization, could be detected by RIG-I and MITA/STING, respectively [19, 20]. The activation of different innate immune signaling pathways may induce type III IFN response and inhibit HBV replication [20].…”

Differential expression of innate immune response genes in clinical phases of chronic hepatitis B infection

“…Cui et al. failed to detect the induction of IFN‐α, IFN‐β, IFN‐λ, TNF‐α and IL‐6 when viral gene expression and replication were clearly being suppressed, and Liu et al. recently revealed that rather than being silent, HBV may be efficient in inducing anti‐/pro‐inflammatory cytokines, but less potent to activate IFN response in patients .…”