HBV DNA is a substrate for the cGAS/STING pathway but is not sensed in infected hepatocytes

Abstract

HBV chronic infection is a critical risk factor for hepatocellular carcinoma. Although debated, the absence of innate immune response to HBV infection in hepatocytes is becoming the current view. However the underlying reasons are poorly understood. This study aims to define potential viral pathogen-associated molecular patterns (PAMPs) and the pattern recognition receptors (PRRs), and to elucidate whether HBV counteracts the innate pathways. The innate immune response to HBV infection was monitored by interferon-stimulated gene 54 (ISG54) mRNA, a direct downstream transcriptional target of Interferon Regulatory Factor 3 (IRF3), or IRF3 phosphorylation. The immunostimulatory potential of naked HBV DNAs or RNAs and the respective PRRs were determined upon viral nucleic acid transfection in immunocompetent cells including knockout cells lacking key molecules of innate pathways. The expression and functionality of DNA and RNA sensing pathways in primary human hepatocytes (PHH) were assessed. The inhibition of the DNA-sensing pathway by HBV was tested using IRF3 nuclear translocation assay. Our study revealed that HBV infection does not induce an innate response in infected hepatocytes, even in absence of HBV X protein. HBV relaxed-circular DNA (rcDNA) and DNA replication intermediates, but not HBV RNAs, are immunostimulatory and sensed by Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase (cGAS) and Stimulator of Interferon Genes (STING). Although PHH express DNA sensors to reduced levels compared to myeloid cells, they can respond to naked HBV rcDNA. However, we show that the absence of innate response to HBV infection in hepatocytes is not due to an active inhibition of the DNA sensing pathway by the virus. HBV passively evades the innate immune response in infected hepatocytes by (i) producing non-immunostimulatory RNAs, (ii) avoiding sensing of its DNAs by cGAS/STING without active inhibition of the pathway, possibly through shielding of the viral DNAs by the capsid. Author summaryInnate immune responses are the first line of defense against viral infections. They lead to the production of antiviral factors after recognition of specific viral features by the infected cells. Here we show that HBV, a major cause of liver cirrhosis and cancer, avoids recognition by infected hepatocytes through different means. First, HBV RNAs, contrarily to other viral RNAs, are not immunostimulatory. Second, we show that naked HBV DNAs are recognized by cGAS/STING and induce an innate immune response. Furthermore, we demonstrate that this pathway is active in hepatocytes and is not inhibited by the virus. Instead, we propose that HBV DNAs are not accessible to cGAS/STING in the context of an infection. This might be due to shielding of the viral DNA by the viral capsid.