The Molecular Mechanism Of Host Proteins RNF114 And PSMB1 Regulating PRRSV Replication

Porcine reproductive and respiratory syndrome(PRRS)has been a major threat to the pig industry worldwide since its outbreak in the late 1980's.In 2006,the outbreak of highly pathogenic PRRS in China severely hit the pig industry in China.With the variation and evolution of PRRSV,it is an urgent problem to find effective and sustainable anti-PRRSV strategies.The function of PRRSV nonstructural protein 12(Nsp12),which might play essential roles in viral replication and production,remains unknown.In this study,we identified two host proteins,RNF114 and PSMB1,which can inhibit PRRSV replication through degrading viral Nsp12.RING finger protein 114(RNF114),also known as zinc finger protein 313,belongs to the family of RING-domain E3 ligases(RING E3s),which plays an important regulatory role in the innate immune response and host–pathogen interaction networks.The expression level of PRRSV nucleocapsid(N)protein,the viral titer in the supernatant and viral RNA copy number in cells were significantly reduced in MARC-145 cells overexpressing RNF114 compared with those in control cells,whereas RNF114 knockdown increased viral titer and N protein expression.indicating that RNF114 could inhibit the replication of PRRSV.In addition,we found that PPRSV infection led to increased RNF114 levels during the middle and late phases of infection in both porcine alveolar macrophages and MARC-145 cells.The screening between RNF114 and PRRSV Nsps showed that RNF114 only interacted with Nsp12.RNF114 reduced Nsp12 protein levels in a dose-dependent manner,which was associated with its E3 ligase activity.RNF114 involves the K27-linked polyubiquitination of PRRSV Nsp12,which promotes the degradation of Nsp12 by the proteasome-dependent pathway and thus inhibits PRRSV replication.PSMB1 is a member of the proteasome subunit(PSMBs)family,which regulates the innate immune response during viral infection.In this study,we demonstrated that PSMB1 can inhibit the replication of PRRSV.Screening between PSMB1 and PRRSV Nsps by Co-IP assay showed that PSMB1 interacted with Nsp12.In addition,we found that Nsp12 could be degraded by PSMB1 in a dose-dependent manner.The proteasome inhibitors and autophagy inhibitors assays showed that the degradation of Nsp12 by PSMB1 was regulated by autophagosomal pathway.Co-transfection of PSMB1 and Nsp12 increased the level of intracellular autophagy and both of them co-located in lysosomes.Moreover,we found that the selective autophagy cargo receptor protein NBR1 interacted with PSMB1 and Nsp12 by Co-IP assay,which involved the autophagy degradation process of Nsp12.Furthermore,our results showed that the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 Lys-130.In conclusion,these results indicate for the first time that PSMB1 is an anti-PRRSV host protein and it inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway.In summary,we identified two host proteins RNF114 and PSMB1 as anti-PRRSV factors in this study,and the mechanism was elucidated experimentally.Our study not only highlights the importance of host factors in PRRSV replication,but also suggests potential antiviral therapies.