Inhibition Of Hepatitis B Virus Replication By Activation Of The CGAS-STING Pathway

Hepatitis B Virus (HBV) is a small DNA virus, which is a major health problem of the world especially in China. The infection of HBV can cause chronic infection, which may further lead to liver failure with cirrhosis and hepatocellular carcinoma (HCC). HBV contains a 3.2 kb-sized, partially double-stranded genomic DNA (relaxed circular DNA, RC-DNA), which will be converted into a plasmid-like covalently closed circular DNA (cccDNA) inside the host cell nucleus during infection, and serves as the template of the synthesis of pregenomic RNA (pgRNA) and other mRNAs.It is reported that a newly discovered cytosol DNA sensor cGAS can directly bind with the foreign DNA in cytoplasm and can defense the infection of HIV and other viruses by activating STING to stimulate the expression of IFN-β.When activated by virus DNA, cGAS go through large conformational changes, opens up the catalytic pocket for ATP and GTP binding and subsequent cGAMP synthesis. cGAMP is a second messenger which can bind with STING to trigger the downstream signaling pathway that produces type I interferon and other cytokines. Before this study, whether cGAS could recognise the infection of HBV or the effects of the cGAS-STING pathway to HBV replication remains unknown.In this research, we studied the effects of the cGAS-STING pathway to HBV replication by a series of experiments on cell lines, human peripheral blood mononuclear cells (PBMCs), and a hydrodynamics-based mice model. We found that the over expression of cGAS and STING were needed in activating this signaling pathway, and the DNA recognition by cGAS was not sequence-specific. Furthermore, both the two types of cGAMP could activate the cGAS-STING pathway dependent 1FN-β promoter. Then, we investigate whether activation of the cGAS-STING pathway could affect HBV replication by cotransfected an HBV replication-competent plasmid (pHBV1.3) in HepG2 cells, and found that the activation of cGAS-STING pathway could suppress the synthesis of virus RNA, DNA, and gene expression of HBV in HepG2 cells and L02 cells, while loss function of cGAS had no such inhibition effects. Next, the experiments on human peripheral blood mononuclear cells (PBMCs) showed that the knockdown of endogenous cGAS leads to a higher level of intracellular HBV DNA level in cytoplasm, which suggests that the cGAS-STING pathway could defend the infection of HBV in PBMCs. By testing the effects on four promoters of HBV by the activation of cGAS-STINGS pathway, the results showed that the activity of EnhⅡ/C (nt 1415-1815), SP1 (nt 2707-2849), SP2 (nt 2937-3182) were declined, while the activity of EnhⅠ/X (nt 950-1375) changed little, what’s more, we found that the cGAS-STING pathway have no effect on the stability of HBV pgRNA. The experiments on a hydrodynamics-based mice model showed that the cGAS-STING pathway could inhibit HBV gene expression and replication in vivo. Meanwhile, we found that cGAMP, the product of cGAS, could inhibit the replication of HBV through activating STING, this finding suggests that cGAMP could be developed as potential inhibitors to HBV resplication. What’s more, our study showed that the cGAS-STING pathway had no effects on the NF-κB signaling pathway, and the expression level of cGAS could not be markedly enhanced by type Ⅰ IFNs stimulation in HepG2 cells.Taken all the studies together, it’s believed that the cGAS-STING pathway plays a role in the surveillance of HBV infection. Therefore, this study may offer a novel strategie for anti-HBV drug developments.