Exploration Of Nsp12 Function In The PRRSV Life Cycle

Porcine reproductive and respiratory syndrome(PRRS)is caused by porcine reproductive and respiratory syndrome virus(PRRSV)and threatens the pig industry seriously.From the late 1980 s to the early 1990 s,the disease first broke out in the United States and Europe.The mutation and the recombination rate of PRRSV is so high that poses a great challenge for us to prevent and control of it.Studying and understanding the biological characteristics of PRRSV is of great significance for the prevention and control of PRRSV.Based on bioinformatics predictions and experiments,14 nonstructural proteins were encoded in the region of ORF1 a and ORF1 b of PRRSV.Among them,Nsp1 is associated with the synthesis of PRRSV subgenomic m RNAs.Nsp2 and Nsp3 are involved in the formation of replication-associated complex double membrane vesicles(DMVs).Current research suggests that PRRSV replication is performed on DMVs.Nsp4 encodes a 3C-like serine protease.In addition,Nsp1?/?,Nsp2,Nsp4,and Nsp11 have shown to be associated with the inhibition of innate immune signaling pathways.ORF1 b region encodes 4 nonstructural proteins(Nsp9 to Nsp12).Nsp9 is an RNA-dependent RNA polymerase(Rd Rp)and Nsp10 is an RNA helicase.Related reports have confirmed that Nsp9 and Nsp10 are Pathogenic-related genes of highly pathogenic PRRSV.Nsp11 has endonuclease and deubiquitinase activity.In addition,Nsp11 has the function of inhibiting NLRP3 inflammasome and regulating cell life cycle.In the entire ORF1 b region,only the function of Nsp12 is not yet clear,and almost no relevant reports has been reported.Nsp12 contains 153 amino acids and the molecular weight is about 17 kDa.It locates at the end of the ORF1 b region and can increase the expression of IL-1? and IL-8.In addition,HSP70 is a relatively well-defined protein that can interact with Nsp12.Inhibition of HSP70 expression can significantly reduce viral mRNA synthesis and virus replication.In this study,the effects of Nsp12 on virus replication and the related biochemical characteristics were studied.We analyzed the conserved Nsp12 sequence in different PRRSV strains and construct Nsp12 deletion mutants based on virus infectious clone to investigate the effect of replication;We also explore Nsp12 dimerization phenomenon and its two disulfide bonds.On the basis of the replicating subsystem constructed in our laboratory,the related research on whether Nsp12 participates in the synthesis of virus RNA is performed.The results showed that the amino acid sequence of Nsp12 among different PRRSV strains were highly conserved,and Nsp12 deletion failed to rescue the virus indicating that Nsp12 was crucial for PRRSV replication.Biochemical analysis showed that Nsp12 was easily oxidized to form dimers and polymers.Both the dimerization of Nsp12 and N protein were caused by oxidation.Unlike N protein,Nsp12 was not pakaged in virus particles.Analysis of three cysteine mutants in Nsp12 indicated combination of 35 th and 79 th cysteine mutants fail to rescue the virus,this indicated that 35 th and 79 th cysteine are essential for the replication of PRRSV.Finally,we developed a mini-genome system that uses firefly luciferase as a reporter to evaluate the PRRSV negative strand synthesis,and we demonstrated that combinations of open reading frames 1a(ORF1a)and ORF1 b are necessary for viral minus-strand RNA synthesis.Basedon this system,we also demonstrated that Nsp12 has been involved in the synthesis of virus RNA.This study explored the biochemical characteristics and functions of Nsp12,which will lay a foundation for exploring the biological function of the protein.