BHV-1 UL41 Exerts VHS Function In CGAS-STING Signaling Pathway And Virus Replication

Bovine herpesvirus type I(BHV-1)is an important pathogen causing bovine respiratory disease syndrome.BHV-1 can infect cattle of all ages,occasionally naturally infected with other animals,also known as infectious bovine rhinotracheitis virus,can cause a variety of respiratory and reproductive system diseases in cattle.The prevalence and infection of BHV-1 cause huge economic losses to the cattle industry in China and even worldwide,and is essential for the prevention and control of the disease and the study of pathogen latency mechanisms.The viral host shutoff protein(VHS)encoded by BHV-1 UL41 gene is a tegument protein,which is transcribed and expressed at the late stage of the virus infections.VHS has a similar substrate activity to RNase A,which can break and degrade m RNA molecules by binding to the UTR region of viral and host m RNA to inhibit m RNA transcription and expression.Therefore,it has a certain regulatory effect on virus replication,and also has an inhibitory effect on the host’s antiviral innate immune pathway.The innate immune system monitors extracellular and extracellular danger signals through pattern recognition receptors and mounts an immune response against pathogens or self-tissue damage.Cyclic GMP-AMP Synthase(cGAS)is an important member of the nucleotidyltransferase family.After cGAS recognizes and binds to pathogen DNA,it can catalyze adenosine triphosphate and guanosine triphosphate to synthesize the second messenger 2’3’-c GAMP,2’3’-c GAMP can activate the membrane protein interferon gene stimulator(STING)on the endoplasmic ret iculum to regulate innate immune function,by intercalating STING in the depression formed by polymerization,it performs functions such as recruitment and activation.Activated STING recruits TBK1 and NF-κB,activates TBK1 which in turn induces IRF3 clustering and phosphorylation,or activates p65 through the NF-κB pathway.Activated IRF3 and p65 molecules enter the nucleus,transmit information and initiate the activation of the interferon(IFN)pathway,induce the production of antiviral immune molecules such as interferon and its downstream such as myxovirus resistant protein(Mx)1 and viperin,and then play a host antiviral role.In recent years,studies between the cGAS-STING signaling pathway and DNA virus infection have attracted much attention.In this study,we investigated the mechanism of negative regulation of cGAS-STING signaling pathway and the effect on viral replication with the function exploring of BHV-1 UL41 encoded VHS.In the study of the negative regulation of cGAS-STING signaling pathway by BHV-1 VHS,the mechanism of DNA molecule recognition by bovine cGAS-STING to initiate IFN antiviral innate immune pathway was first studied,and the obtained sequences were analyzed for biological characteristics,homologous evolutionary analysis and protein three-dimensional structure prediction by cloning bovine cGAS and STING.Eukaryotic vectors expressing bovine cGAS and STING fusion EGFP protein were constructed,and their cellular sublocalization was analyzed by organelle tracking dyes.The experiment further determined that cGAS had the ability to synthesize2’3’-c GAMP,and the dual luciferase reporter vector assay system showed that cGAS-STING could activate IFN-β,IRF3,and NF-κB promoter activity,while Western blot analysis demonstrated that cGAS-STING could phosphorylate TBK1 and p65 molecules,important members of the pathway.In BL cells stimulated by poly(d A:d T),poly(I:C),BHV-1 and BPIV3,the significant activation of IFN-β,Mx1 and Viperin by poly(d A:d T)was inhibited by cGAS and STING inhibitors,and BHV-1 showed some negative regulation in this pathway.The above results showed that bovine cGAS had the ability to synthesize 2’3’-c GAMP,and then initiated bovine STING-dependent innate immune regulatory pathways and induced the expression of IFN and downstream related antiviral immune molecules to achieve antiviral effects.In order to explore the mechanism by which BHV-1 VHS negatively regulates the antiviral innate immune pathway,the cell model was screened by establishing the cGAS-STING pathway,and the dual luciferase reporter vector analysis screened out the inhibitory effect of BHV-1 UL41on IFN-β,IRF3 and NF-κB promoter activation.Then,the effect of UL41 on IFN-βactivation pathway was analyzed by Seq-RNA,and the regulation of UL41 on downstream molecules of IFN pathway was analyzed from differential gene expression,differential gene GO enrichment,and KEGG pathway enrichment.The results revealed that UL41 had extensive regulatory effects on the antiviral innate immune pathway of IFN-βproduction.Western blot analysis of UL41 significantly inhibited the overexpression of cGAS,STING and Viperin,promoted the expression of Mx1 protein under BHV-1 infection and activated by IFN-β,and inhibited the expression of Viperin induced by IFN-β.In order to further explore the mechanism by which UL41 negatively regulates the cGAS-STING signaling pathway,we designed cGAS,STING and Viperin UTR sequence recognition primers with reference to the sequences published by NCBI,and analyzed the UTR region specifically bound by UL41 with RIP analysis.Real-time quantitative PCR determination of UTR transcriptional degradation.The above results indicate d that BHV-1 UL41 negatively regulated the cGAS-STING-mediated DNA recognition and antiviral innate immune signaling pathway through the mechanism of binding and degrading cGAS,STING,Viperin UTRs region.Finally,the UL41 sequence was analyzed,and three groups of sg RNAs with relatively high scores were designed according to the sg RNA online prediction software,which were connected to the p X330 CRISPR/Cas9 system editing vector carrying Cas9 protein,resp ectively,and the sg RNAs introduced into the cells guided the precise localization of Cas9 protein to cut the BHV-1 genome,and the UL41 gene was mutated by non-homologous recombination technology.Pure BHV-1 UL41~-deficient strains were obtained by plaque purification technique and Western blot screening.Finally,BHV-1 UL41 expression silenced strain A2 was obtained by sequencing and Western blot analysis,and later named BHV-1 UL41~-.The one-step growth curve,physicochemical properties and cell tropism of the recombinant BHV-1 UL41~-strain and the parental strain(WT)were studied by measuring the virus titer,and the results showed that the BHV-1 UL41~-strain replicated earlier than the WT and slowed the virus replication cycle;the BHV-1 UL41~-strain showed stronger acid sensitivity and temperature sensitivity;and the selection of cell tropism by the BHV-1 UL41~-strain and the WT showed consistency.Compared with the parental strain,BHV-1 UL41~-strain showed increased transcription of immediate early and early genes,decreased transcription of late genes,and increased transcription of innate immune-related factors by real-time PCR analysis.Baculovirus multi-cell delivery system achieved delivery of BHV-1 UL41 gene on MDBK cells,and UL41significantly inhibited viral replication at 6,12 and 24 h of BHV-1 infection in the overexpression UL41 study system.The above results showed that BHV-1 UL41 is a non-essential gene for BHV-1 replication,which can affect viral replication and parti cipate in the regulation of transcription of antiviral innate immune-related factors.In this study,we demonstrated that BHV-1 UL41 exerts VHS functions in cGAS-STING signaling pathways and viral replication.Bovine has a functional cGAS-STING mediated DNA recognition signaling pathway,and BHV-1 infection negatively regulates this pathway.UL41inhibits cGAS-STING mediated IRF3,NF-κB,and IFN-βpromoter activation and negatively regulates the cGAS-STING-IFN-βaxis and downstream immune molecules of the pathway,specifically BHV-1 UL41 binds and degrades the cGAS,STING,viperin AREs,and STING m RNA5’cap region.Overexpression of UL41 inhibited replication of BHV-1,immediate early and early gene transcription and promoted late gene transcription in the e arly stage of reinfection.The BHV-1 UL41~-strain showed features of an earlier and slower viral replication than WT and was more sensitive to high temperatures and acidic environments.This study provides a scientific theoretical basis for exploring the mechanism of negative regulation of IFN antiviral innate immune pathway by BHV-1 UL41 and points out a research direction for BHV-1 disease prevention and control.