Study On The Effect Of Porcine TRIM21 On PRV Replication And Its Molecular Mechanism

Pseudorabies Virus(PRV),belongs to the genus Varicelloviru,Alphaherpesvirinae subfamily within the family Herpesviridae,and is a double-strand linear DNA with 143 kb and can encode more than 70 proteins.Like other members of the varicella virus,PRV is neurotropic and can establish latent infection in the peripheral nervous system of the infected animal.As the natural reservoir of PRV,pigs exhibit neurological impairment in newborn piglets and reproductive failure in sows after the PRV infection.Although the pig is the sole host for PRV viruses,it can infect many animals,including sheep,dogs,foxes,tigers,bears,etc.More than 20 cases of severe pseudorabies encephalitis in humans have recently been reported.All of these patients infected with PRV variants had previous close contact with pigs,suggesting that swine may be a pathogenic source of PRV infection in humans.Given the significant impact of PRV on aquaculture and human health,in-depth exploration of the molecular mechanisms of PRV infection is of great practical significance for animal disease prevention and control,as well as for safeguarding people’s health.Tripartite motif proteins(TRIMs)belong to the E3 ubiquitin ligase family and are found in almost all multicellular animals.TRIMs have more than 80 members in humans and have multiple functions involving most life activities,including proliferation,differentiation,apoptosis,gene expression,signal transmission,damage repair,inflammation,and immunity.The amino terminal of TRIM protein contains three relatively conservative domains(RBCC),including Really Interesting New Gene(RING)domain,the B-BOX domain,and the coiled-coil domain(CC).The C-terminal of most TRIMs contains the PRY/SPRY domain,which can mediate the interaction between TRIMs and RNA or protein.TRIM21 was first discovered as an autoantigen in patients with autoimmune diseases.Later,it was found that TRIM21 plays a crucial role in regulating the signal pathway of type I interferon during virus infection.In this study,we first characterized the sequence and structural features of porcine TRIM21.The results showed that the homology between porcine TRIM21 and TRIM21 of 19 species was more than 80%(Table 1),including Balaenoptera musculus(Gen Bank XP＿036718516.1),Balaenoptera acutorostrata scammoni(XP＿007173691.1)etc.Porcine TRIM21 has the highest homology with TRIM21 from B.musculus and B.acutorostrata scammoni,with 83.8%.Furthermore,PK-15-TRIM21 overexpression/knockout cell lines were constructed to examine the effect of TRIM21 on the proliferation of PRV,and the results showed that TRIM21 inhibited PRV replication.We further examined IFN expression after PRV infection of TRIM21 overexpressing/knockout cells and showed that IFNα was suppressed after TRIM21 overexpression,whereas TRIM21 knockdown was resistant to IFNγ was significantly promoted.Moreover,IFNs and proinflammatory factors are essential in innate immune and antiviral responses.The impact of TRIM21 on proinflammatory aspects continues to be explored.We found that after PRV infection,TRIM21 suppressed proinflammatory IL-6 and TNF α.The IE180 protein is the only immediate-early transcriptional activator of the PRV virus and plays a crucial role in translating the viral genome.To further explore the molecular mechanism by which TRIM21 inhibits PRV proliferation,the gene expression profile of PRV viral immediate-early protein IE180 was further examined by fluorescence quantitative PCR and immunoblotting.The results indicated that TRIM21 affected the expression of the immediate early protein IE180,and we speculated that TRIM21 interacted with IE180 of the PRV virus.By constructing a TRIM21 domain deletion mutant to explore the critical domain of trim21 involved in the interaction with PRV,we found that deleting the PRY/SPRY domain in TRIM21 eliminates the inhibition of TRIM21 on PRV and increases IFN α Expression.The expression of IFNβ/γ has not significantly difference.At the same time,deleting the PRY/SPRY domain improves the inhibitory effect of TRIM21 on IL-6,TNF-α,and IE180.The above results indicate that PRY/SPRY is a key domain for TRIM21 to inhibit PRV replication.In summary,this study shows that the homology between pig TRIM21 and 19 species of TRIM21 is over 80%,and the domain similarity between pig TRIM21 and B.musculus and B.acutorostrata scammoni TRIM21 is relatively high,especially in the RING and B-Box domains.Overexpression of TRIM21 inhibits PRV proliferation and the expression of the immediate early gene IE180,as well as IFNα、 IL-6、TNF-α.The deletion of the PRY/SPRY domain in TRIM21 eliminated TRIM21’s inhibition of PRV and its immediate early gene IE180 expression,and eliminated TRIM21’s inhibitory effect on IFN-α、 IL-6、TNF-α.These results indicate that the PRY/SPRY domain is a key domain for TRIM21 to inhibit PRV replication.