The Mechanisms Of Pseudorabies Virus Tegument Protein UL13 In Regulation Of Host Antiviral Immunity

Pseudorabies virus（PRV）infection in swine generally induces a virulent infectious disease that leading to significant economical losses in the swine industry.Since 2011,the emergence of variant PRV strains in China exacerbated the severity of the diseases.Besides pigs,PRV infection can also cause diseases or death in a variety of domestic animals,dogs,cats,laboratory animals and wild animals cause disease or death.Moreover,recent studies have shown that PRV can directly infect humans,which leads to severe damage in nervous and respiratory systems,indicating that PRV is a zoonotic virus.Therefore,it is of great significance to investigate the pathogenic mechanisms of PRV.It has been suggested that herpesviruses can employ multiple strategies to antagonize host innate antiviral responses for immune evasion.Among them,tegument proteins play critical roles in regulation of host immune innate responses.cGAS is the major cytosolic DNA sensor that recognizes viral DNA to initiate STING-dependent innate antiviral responses.However,it is unknown whether and how PRV targets the cGAS-STING axis for immune evasion.To investigate the role of PRV tegument proteins in the regulation of host antiviral immune responses,9 key PRV tegument proteins were expressed in HEK293 T cells and screened the effects of those viral proteins on activity of IFN-β and ISRE promoters via luciferase assays.Of note,it was identified UL13 and US3,two protein kinases of PRV,dramatically impaired the activity of IFN-β promoters upon B-DNA stimulation.It focused on UL13 as its role in regulation of host innate immune responses were not reported previously.Immunoblotting analysis showed that UL13 expression deceased STING protein levels without affecting cGAS protein expression,an upstream adaptor protein of STING.As a result,UL13 suppressed activation of STING-mediated antiviral immune responses.Consistently,knockdown or deletion of UL13 enhanced host antiviral immune response mediated by DNA viral infection,indicating that UL13 is a negative regulator of host antiviral immunity.In addition,UL13-deficient PRV showed less pathogenicity in vivo in a murine model than that of wild PRV.Together,these data suggested that PRV tegument protein UL13 negatively regulates host antiviral immune responses via targeting STING-mediated signaling pathway.Mechanistically,UL13 could directly interact with STING via the CDN domain of STING.Further analysis showed that UL13 did not regulate expression of STING m RNA but promote STING degradation via the proteasome pathway.Immunoprecipitation assay revealed that UL13 did not induce STING degradation through the classical K48-linked ubiquitination.Instead,UL13 promoted STING degradation via K27/K29-linked ubiquitination,an unappreciated way previously for STING protein regulation.Finally,we identified that UL13 recruited the E3 ligase ring-finger protein 5（RNF5）to induce K27/K29-linked ubiquitination of STING by mass spectrometry and immunoprecipitation assays.In line with this notion,RNF5 deficiency significantly enhanced host antiviral immune responses in response to PRV infection.In summary,it has identified that PRV UL13 as a novel negative regulator of host innate immunity via targeting STING-mediated immune responses through recruitment of E3 ligase RNF5 to promote degradation of STING protein.These results thus suggest a new function of PRV tegument protein UL13 and provide a potential mechanism for PRV immune escape.Therefore,UL13 might be a promising target for development of novel live vaccines or antiviral drugs for PRV.