The Mechanism By Which Pseudorabies Virus Regulates The DNA Damage Response And CGAS-STING Signaling Pathway In Host Cells

Pseudorabies virus(PRV),the pathogen of Aujeszky’s disease(AD),has caused huge economic losses to the global swine industry.New variant strain is continuing to appear with the evolution of PRV.Especially since 2011,the outbreaks of PR caused by PRV variant strain have happened in many Bartha-K61-vaccinated swine farms in China.The replication of PRV can cause the accumulation of viral DNA in host cells.However,whether these DNA can be sensed by cellular DNA sensors and activate downstream responses,especially DNA damage response and cGAS mediated innate immune response and whether PRV infection can modulate these pathways to benefit its own replication are still unknown.Therefore,it is of great importance to analyze the genetic characteristics of PRV variant and explore the interaction between PRV infection with DNA damage response and cGAS mediated innate immune response for the vaccine development of PRV and understanding the interaction between PRV with host cells.Based on the above backgrounds,this study includes the following three parts:1.Phylogenetic study of PRV variantIn 2018,two field PRV strains,designated JSY7 and JSY13,were isolated from the brain of the pig vaccinated with Bartha-K61 vaccine in Jiangsu Province in our lab and they showed different biological characteristics.To further explore the genetic characteristics of these two PRV field strains,the complete genome sequences of them were sequenced using de novo method The results showed that the JS Y7 genome was comprised of 144,315 bp and the GC content was 73.71%,and the JSY13 genome was comprised of 143,859 bp and the GC content was 73.75%.Alignment of gE amino acid sequences showed that compared with foreign strains there were two aspartic acid insertions occurred at position of 48 and 497 in gE protein of JSY7 and JSY13.The phylogenetic tree based on the complete genome sequences showed that JSY7 and JSY13 were in the same clade with other strains isolated in China.What’s more,it was found that JSY7 had a much closer relationship to the epidemic strains that were isolated after 2011,while JSY13 was much closer to the earlier isolates of China.Phylogenetic tree of gC gene showed that PRV could be divided into two genotypes:genotype Ⅰ was mainly composed of European and American strains and genotype Ⅱ was mainly composed of Chinese strains.In addition,genotype Ⅱ could be further divided into 3 lineages.This study found that JSY7 belonged to lineage 3,while JSY13 belonged to genotype Ⅰ and was divided into the same branch with Bartha.Phylogenetic trees based on the main immunogenic proteins of gB and gD genes showed that JSY7 and JSY13 were located at the same branch with the current epidemic strains,but in different clades with the Bartha strains.Comprehensive analysis of above phylogenetic trees,the result showed that the Bartha strain was phylogeneticly far from the currently epidemic strain,which might be the reason why the Bartha-K61 vaccine could not provide complete protection to PRV variant.Based on above analysises,we also suspected that JSY13 might be a recombinant between JSY7 field strain and Bartha vaccine strain.Simplot software was used to analyze the similarity of the complete genome sequences of these three strains.The result showed that there were three regions of Bartha in JSY13 sequences and the rest parts were in a high similarity with JSY7.Finally,to compared the virulence of JSY7 and JSY13,we infected these two strain of viruses on mice.The survival curve showed that JSY7 was more virulent than JSY13.Taken together,this study comprehensively analized the genetic characteristics of the two field strain JSY7 and JSY13,and also found that JSY13 might be the natural recombination of JSY7 variant strain and Bartha vaccine strain.2.The interaction between PRV infection and host DNA damage responseDNA damage response system was consisted of DNA sensor,tranducer,and effector.Previous reports have showed that virus infection can activate the DNA damage response and regulate the replication of virus.However,the interaction between PRV infection and the host DNA damage response is still unknown.γH2AX was used as the marker of DNA damage response to check whether PRV infection could activate DNA damage response.The results showed that PRV infection could increase the level of yH2AX.What’s more,it was also found that PRV infection could activate both ATM and DNA-PK pathways.To investigate the effect of different DNA damage response pathway on the replication of PRV,Vero-E6 cells were pretreated with specific inhibitors of ATM,ATR,and DNA-PK pathways before PRV infection.The results showed that ATM inhibitor KU55933 could inhibit PRV replication while ATR inhibitor VE-821 and DNA-PK inhibitor NU7441 had no effect on PRV replication.We also used siRNA to knock down ATM,Chk2,DNA-PK,and H2AX to check their role in PRV replication,the results showed that PRV replication was inhibited when ATM,Chk2,and H2AX were knocked down.This study also found that the PRV infection can induce the accumulation of ROS and PRV replication was inhibited when cells were pretreated with ROS inhibitors Apocynin(APO)and Diphenyleneiodonium(DPI).To explore whether there is single PRV ORF that can regulate the level of γH2AX,50 ORFs of PRV were overespressed and the data showed that UL13 kinase protein could increase the level of γH2AX.The kinase dead mutants that were constructed using sitedirected mutagenesis could not increase the level of γH2AX.Finally,UL13 deletion viruses were constructed by using CRISPR/Cas9 system to check whether same effect was shown upon virus infection.The data showed that PRVΔUL13 mutants showed a lower ability in increasing the level of γH2AX than their parent strain JSY13.In summary,this study found that PRV infection could activate ATM pathway mediated DDR which was induced by the accumulation of ROS,and this activation process could facilitate the replication of PRV.We also found that PRV UL13 kinase protein could increase the level of γH2AX and the virulence of PRV in a kinase activity dependent manner.3.PRV UL13 inhibits cGAS-STING pathway mediated IFN-β production by phosphorylating IRF3As a cytoplasmic DNA sensor,cGAS can sense invading viral DNA,catalyze the synthesis of the second messenger molecule cGAMP,activate downstream factor STING,TBK1 and IRF3 to induce the production of IFN-β.A dual luciferase system of the activation of IFN-β promoter mediated by cGAS-STING pathway was constructed to check the effects of PRV ORFs.Through screening 50 PRV ORFs,it was found that UL13 kinase protein could significantly inhibit cGAS-STING-mediated IFN-β transactivation.It was also found that UL13 could inhibit Poly(I:C)and VSV-induced IFN-β activation.Furthermore,it was also found that IRF3 was the target of UL13 and UL13 kinase protein could phosphorylate IRF3 in a kinase-dependent manner.To explore the specific mechanism of UL13 on inhibition of IRF3 transcription activity,non-denature Western-blot was firstly used to check whether UL13 could influence the dimer formation of IRF3.The data showed that UL13 kinase protein could not affect the dimer formation of IRF3 induced by VSV infection and Poly(I:C)treatment.Through IFA experiment,it was found that UL13 could not affect Poly(I:C)induced IRF3 nuclear translocation.This study also found that UL13 could not affect the binding between CBP and IRF3.Next,through chromatin immunoprecipitation experiment,we found that UL13 could significantly inhibit the binding of IRF3 to the IFN-β promoter regulation sequence.Through semi-RT-PCR,we also found that UL13 could inhibit the mRNA level of a variety of ISGs that were induced by cGAS-STING and Poly(I:C).Finally,compared with JSY13,PRVOUL13 could trigger a higher level of IFN-βtransactivation.In summary,we found that UL13 could inhibit cGAS-STING induced IFN-β activation through phosphorylating IRF3.In conclusion,this study firstly expleored the phylogenetic characteristics of PRV,and it was found that JSY13 field strain is a recombinant strain between the Bartha-K61 vaccine strain and JSY7 field strain.Secondly,this stdudy explored the interaction between DNA damage response and PRV infection.It was found that PRV infection can activate DNA damage response,and ATM pathway mediated DNA damage response is required for efficient replication of PRV.In addition,this study also found that PRV UL13 kinase protein can activate DNA damage marker yH2AX in a kinase-dependent manner.Finally,this study explored the regulation of cGAS-STING pathway mediated innate immune response unpon different ORFs(open reading frames)of PRV.It was demonstrated that PRV UL13 kinase protein can inhibit IFN-β production by phosphorylating IRF3.