The Structure And Function Research Of PRRSV Endoribonuclease Nsp11

Porcine reproductive and respiratory syndrome virus(PRRSV) is a member of the family Arteriviridae, order Nidovirales. PRRSV is the causative agent of porcine reproductive and respiratory syndrome(PRRS), which has become one of the most important infectious diseases in the swine industry worldwide. Since the first Chinese PRRSV was isolated in 1995, PRRSV has caused enormous damage in China, especially the outbreak of highly pathogenic PRRSV(HP-PRRSV) in 2006 and reemergence in 2009-2010. Due to the PRRSV genome during the continuous variation, currently effective prevention and control of PRRSV is very difficult for lack of effective vaccines and drugs. PRRSV nsp11 is endoribonuclease which was coded by the virus itself. Nsp11 possesses nidovirus uridylate-specific endoribonuclease(NendoU) activity, which is important for arterivirus replication. In this study, we successfully resolved the first crystal structure of the arterivirus nsp11, and elucidate the dimerization-dependent mechanism drives the NendoU′function. Our results will provide a new route for the development of effective antiviral drugs. The detailed experimental results were shown as follows: 1. Epidemic investigation and molecular variation analysis of PRRSV from thepig breeding farms in hubei provinceWe analyze the epidemiology of PRRSV from 14 pig breeding farms in Hubei province, which indicated that among 668 serum samples, 35(5.24%) were positive for PRRSV detection. Of the PRRSV-positive samples, 13(1.95%) were positive for the C-PRRSV and 24(3.59%) were positive for the HP-PRRSV. The results showed that the HP-PRRSV has become the dominant strain. Meanwhile, the complete nsp2, orf5, and orf7 gene sequences were sequenced and analyzed. The amino acid sequence analysis indicated that the majority of the PRRSV strains contained a common 30-aa deletion at positions 480 and 532-560, similar to the JXA1 strain, when compared with VR-2332. The results indicated seven PRRSV strains collected in this study belonged to HP-PRRSV strain. Meanwhile, the deletion of 59 or 68 aa has been also found, implying that other types of variants may have been prevalent in this region. The phylogenetic analysis on orf5 genes showed that the strains collected during 2009-2010 formed a tightly clustered branch. When compared with the JXA1 strain, they had one mutation(V29→A29) in decoy epitope. Moreover, the number of the potential N-glycosylation sites apparently increased in recent years. Multiple alignments indicated that there were extensive substitutions among orf5 genes. The substitutions(Phe39/Leu39→I39, Leu41→Ser41) in PNE and the increasing of N-glycosylation sites might have allowed these field strains to escape neutralization by the antibodies induced by current vaccines. Finally, the amino acid sequence analysis on orf7 genes indicated that the majority of mutations in Nucleocapsid(N) protein were observed at the residues: Arg11→Lys11, Asp15→Asn15, Lys46→Arg46, Thr91→Ala91, His109→Gln109 and Val117→Ala117.In summary, we investigated the prevalence of PRRSV in the pig breeding farms from hubei region,and analyzed the molecular variation of the epidemic PRRSV strains from 2006 to 2012. It provided theoretical basis for effectively controlling the epidemic of PRRSV in Hubei province. 2. The structure and function research of PRRSV endoribonuclease nsp11The first crystal structure of the arterivirus nsp11 was successfully resolved. The crystal structure of the PRRSV nsp11 exhibits a unique structure and assembles into an asymmetric dimer that is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were relatively conservative compared with other domains, especially in the “active site loop”(His129 to His144) and “supporting loop”(Val162 to Thr179) regions. Moreover, the structural comparison demonstrated that residues His129, His144, Lys173, Thr177, Asp180, Asp204 and Tyr219 from nsp11 superimpose well onto the corresponding residues of coronavirus nsp15, indicating a similar endoribonuclease cleavage mechanism shared among families Arteriviridae and Coronaviridae.PRRSV nsp11 functioning as a dimer rather than a hexamer that is completely different from the coronavirus nsp15. Firstly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Moreover, we analyzed the detailed dimerization interface by PDBePISA, and our biochemical data showed that these mutations(S74A and F76A) significantly disrupt the dimerization in solution and significantly reduced the NendoU activity, indicating that the dimer is the biologically functional unit. In the nsp11 structure, we find that the “active site loop” and “supporting loop” are packed against one another, which is the structural basis for functional nsp11. The structure of the “active site loop” and “supporting loop” is stabilized by monomer-monomer interactions(Hydrogen bond and hydrophobic interaction).The mechanism of the NendoU activity from PRRSVnsp11 was analyzed by biochemical experiments. Firstly, we analyzed the NendoU activity of the wild-type and H129 A, K173 A, T177 A and Y219 A mutant proteins by the FRET assays, which showed that the activity levels of the mutants were significantly reduced compared with the wild-type. Our results indicated that these residues are important NendoU active sites. Besides, structural analysis showed that three putative catalytic residues(His129, His144 and Lys173) surround a positively charged cavity in the catalytic domain, with Thr177 located in the middle of the groove. We find that the NendoU activity of the H129 A mutant is much lower because the His129 is thought to accept protons during cleaves RNA substrate. In addition, Thr177 and Tyr219 could also be important for substrate recognition and binding. Therefore, the catalytic activity levels of the T177 A and Y219 A mutants were significantly decreased.The potential cytotoxicity of PRRSV nsp11 may inhibit IFN-β promoter activation. We find that the overexpression of wild-type nsp11 markedly inhibited the activity of the IFN-β luciferase reporter induced by Sendai virus(SEV), while the mutants(S74A, S76 A, H129 A, K173 A, T177 A and Y219A) partly lost the capacity to block the activation of IFN-β promoter. However, our results showed that the value of pRL-TK, an internal control reporter, was significantly lower in cells expressing wild-type nsp11, indicating that the wild-type nsp11 inhibits host gene expression. Coincidentally, all the tested nsp11 mutants without reducing the value of pRL-TK, did not significantly inhibit IFN-β induction. Therefore, we could not exclude the possibility that the potential cytotoxicity of wild-type nsp11 inhibits IFN-β induction, outside the context of infection. Whether the NendoU′function of nsp11 specifically contributes to the decline of innate immune functions in PRRSV infection requires further investigation.In summary, these findings may help elucidate the mechanism underlying arterivirus replication and may provide great potential for the development of antiviral drugs.