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

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 summary Innate 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.

pathways. The expression and functionality of DNA and RNA sensing pathways in primary 48 human hepatocytes (PHH) were assessed. The inhibition of the DNA-sensing pathway by 49 HBV was tested using IRF3 nuclear translocation assay. 50 Our study revealed that HBV infection does not induce an innate response in infected 51 hepatocytes, even in absence of HBV X protein. HBV relaxed-circular DNA (rcDNA) and 52 DNA replication intermediates, but not HBV RNAs, are immunostimulatory and sensed by 53 Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase (cGAS) and 54 Stimulator of Interferon Genes (STING). Although PHH express DNA sensors to reduced 55 levels compared to myeloid cells, they can respond to naked HBV rcDNA. However, we show 56 that the absence of innate response to HBV infection in hepatocytes is not due to an active 57 inhibition of the DNA sensing pathway by the virus. Innate immunity is the first line of defense against pathogens. Detection of pathogen-75 associated molecular patterns (PAMPs) by cellular pathogen recognition receptors (PRRs) 76 triggers signaling pathways leading to interferon (IFN) production, which induces antiviral 77 interferon-stimulated genes (ISGs) and pro-inflammatory cytokines. 78 In viral infections, viral RNA or DNA are common PAMPs that can be detected by 79 cytosolic PRRs. Members of the RIG-I-like Receptor (RLR) family such as Retinoic Acid 80 MDA5-pathways [3]. Surprisingly, MDA5 was also reported to bind HBV DNA [3]. 106 Recently, sensing of HBV DNA by the cGAS/STING pathway was suggested [17,10] but the 107 expression and functionality of this pathway in hepatocytes is unclear [8,10,13,17,18]. 108 Therefore, the immunostimulatory potential of HBV nucleic acids and the PRR involved 109 require further investigations. 110 HBV could inhibit the innate immune response to escape its antiviral effects, as 111 suggested by several publications [6,10,[19][20][21][22][23][24][25][26][27][28]. In particular, the regulatory Hepatitis B virus 112 X protein (HBx) has been described to inhibit the MAVS pathway [20,26], while the viral  [8,30,31], but the DNA-sensing pathway has been less extensively investigated. 119 Guo et al. [11] proposed that HBV expression does not affect the response to dsDNA or 120 cGAMP stimulation in HepAD38 cells expressing an integrated copy of HBV genome under 121 the control of a tetracycline-controlled promoter, but did not study a genuine HBV infection. 122 Here, we confirmed the lack of innate response to HBV infection in hepatocytes, 123 which was not restored by HBx depletion. To understand the lack of innate response to HBV 124 infection we first assessed the immunostimulatory potential of naked HBV DNAs and RNAs 125 in monocyte-derived dendritic cells (MDDCs) as a model for highly immunocompetent cells. 126 For the first time, we could demonstrate that HBV RNAs are not immunostimulatory. On the 127 contrary, naked HBV rcDNA can elicit a strong innate response, mediated by the 128 cGAS/STING pathway. In hepatocytes, this pathway is expressed at a low level but retains its 129 ability to sense HBV rcDNA. Furthermore, we demonstrate that HBV infection does not 130 inhibit the innate immune response to foreign DNA in hepatocytes. 134 We first determined whether hepatocytes could mount an innate immune response to 135 HBV infection. HepG2-hNTCP cells, overexpressing the viral receptor NTCP, were infected 136 with HBV for 16 days. ISG54 mRNA was chosen as a marker for the innate immune 137 response, as it is a direct target gene of IRF3-dependent transcription [32,33]. IFN-λ1 mRNA 138 expression, previously proposed to be induced by HBV [4,5] was also analyzed (Fig 1). 139 Although the cells were efficiently infected, as demonstrated by the accumulation of HBV 140 RNAs over time, we observed no induction of ISG54 or IFN-λ1 ( Fig 1A). The innate response 141 to HBV infection was next investigated in primary human hepatocytes (PHH) (Fig 1B). HBV    Altogether, our data confirm the absence of an innate response to HBV infection in 157 hepatocytes and rule-out an inhibition of this response by HBx. Furthermore, the absence of 158 IRF3 phosphorylation suggests either an absence of sensing by PRRs or, alternatively, an 159 inhibition of the signaling pathways upstream of IRF3 phosphorylation.   Hepatocytes are competent for DNA sensing 246 We next tested whether the low levels of cGAS/STING correlate with an altered 247 functionality of the pathway in hepatocytes. To this aim, we first stimulated HepG2-hNTCP 248 with the STING agonist cGAMP ( Fig 5A). Interestingly, ISG54 was transiently but including HBV rcDNA, to some extent, but cGAS and STING expressions are limiting. 268 We next assessed the functionality of the DNA sensing pathway in PHH ( Fig 5C). 269 Interestingly, transfection of PHH with undigested, but not with DNAse-digested HBV In this study, we confirmed the absence of an innate immune response to HBV 301 infection in hepatocytes and investigated the reasons for this escape. We first demonstrated 302 that naked HBV RNAs are not immunostimulatory. In contrast, we found that naked HBV 303 DNAs, and particularly the rcDNA from viral particles, can be sensed by the cGAS/STING 304 pathway. We showed that in hepatocytes, including PHH, this pathway is expressed at a low 305 level, but is sufficient to respond to transfected HBV rcDNA. We further demonstrated that 306 HBV does not actively inhibit the innate response to foreign DNA in infected hepatocytes. 307 We observed no induction of ISG54 or IFN-λ1 and no phosphorylation of IRF3 in 308 HBV-infected hepatocytes (Fig 1). These data differ from Shlomai et al. [       were previously described [4]. THP-1 cells were cultivated in RPMI supplemented with 10% fetal calf serum (FCS) and 2 mM L-Glutamine. For differentiation, 40 ng/ml of phorbol 12myristate 13-acetate (PMA) were added for 48 hours.

HBV does not induce an innate immune response in hepatocytes
Cell lines were authentified as described in the Supplementary  Baden-Württemberg Hessen.

HepG2-hNTCP overexpressing cGAS and STING
HepG2-hNTCP were transduced with pLX304-based lentiviral vectors encoding V5tagged cGAS and selected with 20 µg/ml of blasticidin. For each experiment, the selected cells were freshly transduced with pLX304-based lentiviral vectors encoding V5-tagged STING and used 6 days post transduction for experiments.

Other viruses and viral vectors
Sendai virus, strain Cantell, was provided by G. Kochs, Medical Center-University of Freiburg, Germany.
The DNA virus MVA-gfp based on Modified vaccinia Ankara was kindly provided by Gerd Sutter [7,8].

Quantitative reverse transcription PCR (RT-qPCR).
For RT-qPCR analysis, total RNAs were extracted from cell pellets using When indicated, relative change to the control condition was calculated using the formula 2delta delta Ct with delta delta Ct = delta Ct sampledelta Ct control.
For Figure 4, the expression levels of the genes of interest and of 2 reference genes, RPL13A and TATBP, were calculated using a standard curve generated with serial dilutions of THP1 RNA. For each sample, the values of the genes of interest were normalized to the geometric mean of the 2 reference genes. The limit of detection for each sample and each gene of interest was calculated as the RNA amount measured in the last detectable standard dilution of THP1 for the gene of interest divided by the geometric mean of the expression levels of the 2 reference genes for each sample. Anti-rabbit HRP Cell Signaling 7074S

Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 15 min, washed 3 times with PBS, permeabilized with 0,25% Triton-X100 for 7 min at room temperature and blocked for 1 hour in PBS-0,1% Tween (PBST) containing 5% of bovine serum albumine (BSA). The coverslides were then incubated for 2 hours at room temperature with the primary antibodies, washed 3 times in PBST, and incubated for 1 hour with the secondary antibodies (Supplementary Table 4). The coverslides were washed 3 times in PBST and the DNA was stained with Hoechst reagent. Anti IRF3 (rabbit) IRF3 translocation assay, Fig.6 Cell Signaling 11904S D6I4C