Immune Activation Of Human Cervical Epithelial Cells Inhibits HSV-2 Infection And Mechanism Study

Object: Herpes simplex virus type 2(HSV-2),a member of Herpesviridae family,is an enveloped double-stranded DNA virus.HSV-2 commonly causes genital herpes as well as Genital Ulcer Disease(GUD).Infected individuals can carry the virus for life.The human cervical epithelial cells(HCEs)is an important part of the female reproductive tract(FRT)mucosa which participates in the innate immune antiviral response.Studies have shown that human cervical epithelial cells possess TLR3 receptors that can recognize viral doublestranded RNA and produce antiviral factors regulated by interferons upon activation.At present,there are no reports on whether human cervical epithelial cells have functional dsDNA sensors.Therefore,our study examined the expression of DNA/RNA sensors in cervical epithelial cells,and whether these sensors can be activated by Poly(dA:dT),the ligand of DNA sensors,inducing the expression of antiviral factors.Method: The toxicity of Poly(dA:dT)on human cervical epithelial cells(End1/E6E7 cells)was determined by MTT assay andAnnexin V/7-AAD assay.The inhibition of Poly(dA:dT)on HSV-2 infection and replication was detected by real-time PCR and Western Blot.The effect of Poly(dA:dT)on the expression of interferons(IFN-λ1 and IFN-λ2/3)and ISGs(ISG15,ISG56,OAS1,OAS2,MxA,MxB,Viperin and GBP5)in End1/E6E7 cells was detected by real-time PCR,ELISA and Western Blot,and the signal pathway of cellular immunity to recognize Poly(dA:dT)was studied.The influence of Poly(dA:dT)on the expression of DNA/RNA sensors(c GAS,STING,DAI,IFI16,AIM2,DHX29 and RIG-Ⅰ)in human cervical epithelial cells was detected.The RIG-Ⅰ gene was knocked out by the CRISPR/Cas9 technique to determine the mechanism of Poly(dA:dT)inhibiting HSV-2replication.Finally,we constructed the RIG-Ⅰ knockout cells by using CRISPR/Cas9 technology to determine the mechanism by which Poly(dA:dT)inhibits HSV-2 replication.Result: The concentration of Poly(dA:dT)(0.1～10 μg/mL)had no cytotoxic effect on End1/E6E7 cells.Poly(dA:dT)-transfected cells had significantly lower levels of HSV-2RNA,DNA and protein.Mechanistic experiments showed that Poly(dA:dT)could induce the expression of the multiple antiviral factors,such as IFN-λs and ISGs,as well as the phosphorylation of IRFs and STATs proteins.Through studying the effect of Poly(dA:dT)on the expression of DNA/RNA sensors and knocking out the RIG-Ⅰ gene via CRISPR/Cas9 technology,we found that knocking out RIG-Ⅰ gene in the cells resulted in the reduced effect of Poly(dA:dT)on HSV-2 infection and the antiviral factors induction.In addition,RIG-Ⅰ knockout inhibited the activation of IRF/IFN and JAK/STAT pathways induced by Poly(dA:dT).Conclusion: Poly(dA:dT)can activate the DNA/RNA sensors in human cervical epithelial cells and induce the expression of IFN-λs and ISGs,resulting in HSV-2 inhibition.Mechanism studies show that RIG-Ⅰ knockout reduces the activation of the Poly(dA:dT)-induced IRF/IFN and JAK/STAT pathways and Poly(dA:dT)-mediated HSV-2 inhibition.These findings indicate that RIG-Ⅰ plays a key role in Poly(dA:dT)-mediated antiviral immunity.These results provide direct scientific evidence to support potential clinical use of DNA/RNA sensors activators as a treatment option for HSV-2 infection.