The Mechanisms Of Deubiquitinase OTULIN In PRRSV Infection

The ubiquitin-proteasome system played an important role in different stages of viral infection,and viruses have also evolved a variety of strategies for targeting the host ubiquitin-proteasome pathway.In this study,cells infected with porcine reproductive and respiratory syndrome virus(PRRSV)were used to elucidate the new mechanism by which viruses use the ubiquitin-protease system for own proliferation from the perspective of the interaction between viral proteins and deubiquitinating enzymes.(1)Characterization of the interaction between OTULIN expression and PRRSV proliferationThe OTU deubiquitinase with linear linkage specificity(OTULIN)was screened from the transcriptome sequencing data of 3D4/21 cells after PRRSV infection,and the results shown OTULIN expression was up-regulated after PRRSV infection.It was demonstrated that overexpression of OTULIN facilitated the PRRSV proliferation and increased the viral titer of PRRSV.We found that OTULIN overexpression significantly inhibited the the transcriptional levels of interferon pathway-associated TNF-α,IRF3 and NF-κB,and significantly down-regulated the production of IFN-β.(2)OTU domain of OTULIN interacts with PRRSV Nsp11OTULIN interacted with PRRSV Nsp11.Immunofluorescence experiments further demonstrated that OTULIN co-localized with PRRSV Nsp11 and formed complexes during PRRSV infection.Then we mapped the domains of OTULIN and constructed the corresponding truncated bodies,and the results showed the key to binding was the OTU domain.Thereafter,the enzyme active sites on the OTU domain were mutated,which did not affect the interaction between OTULIN and Nsp11,indicating that OTULIN interactd with PRRSV Nsp11 in a non-enzymatically active site.(3)PRRSV Nsp11 interacts with NEMO and over-recruits OTULINIt has been known that OTULIN removed linear ubiquitination targeting NEMO.Further studies have shown that PRRSV Nsp11 interacts with IKKα,IKKβ and NEMO complexes,recruiting OTULIN to excessively remove the linear ubiquitination level targeting NEMO.(4)Superposition removal effect of OTULIN and Nsp11 on type I interferonrelated ubiquitin levelsOTULIN was a specific deubiquitinylating enzyme,while PRRSV Nsp11 specifically removed K48-linked ubiquitin chains.OTULIN and PRRSV Nsp11 had a synergistic reduction effect on cellcular ubiquitin level.Both PRRSV Nsp11 and OTULIN were negative regulators of innate immunity,and they had additive inhibitory effects on NF-κB,IRF3 and IFN-β production in the type I interferon signaling pathway induced by Se V.In addition,the co-expression system of Nsp11 and OTULIN inhibited the nuclear import of P65 more significantly than the single overexpression system of Nsp11 or OTULIN.In summary,our study showed that PRRSV infection up-regulated the expression of the OTULIN,and overexpression of OTULIN contributed to PRRSV proliferation.PRRSV Nsp11 recruitd OTULIN through a non-enzymatic combination,enhancing the ability of OTULIN to remove linear ubiquitination targeting NEMO,resulting in a superimposed effect that inhibitd the production of type I interferons.Our report presented a new model of virus utilization of the ubiquitin-protease system in vivo from the perspective of the viral proteins interacted with cell deubiquitination enzymes,providing new ideas for prevention and control of PRRSV.