| Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens for global pork industry. The nucleocapsid protein (N), an important and highly immunogenic component of virus particles, plays crucial roles in RNA synthesis and virus particles assembly, and its nuclear localization signal (NLS) is inextricably related to the pathogensis of PRRSV. SUMOylation, which targets substrates with small ubiquitin-like modifier (SUMO), presents as an essential theme of post-translational modification in regulating a wide range of cellular processes. Many studies have revealed that the SUMOylation of viral proteins plays an important role in suppressing the host innate immune response, virus immune evasion and regulating virus replication. In this study, the effects of N protein SUMOylation on the virus growth characterization,subcellular distribution of N protein as well as IFN-P production were well investigated, whose results provide many clues for further study on the pathogenesis of PRRSV from the new perspective of post-translational modification, and discovering the anit-PRRSV targets.To identify the SUMOylation of PRRSV proteins, the SUMOylation sites were initially predicted by using bioinformatics softwares such as SUMOSp1.0a, SUMOplot and seeSUMO. One SUMO E2 conjugating enzyme Ubc9 was served as the "bait" protein to screen out the interacted viral protein in yeast two-hybrid assay system. The results showed that Ubc9 could interact with Nsp1β, Nsp4, Nsp9,Nsp10 and N. Then the interaction of Ubc9 with Nsp1β, Nsp4, Nsp9, Nsp10 and N protein in host cells was further confirmed by co-immunoprecipitation (Co-IP), GST pull-down and confocal assays,respectively. In order to further analyze the effects of Ubc9 on viral replication and genomic RNA synthesis in vitro, siRNA and lentivirus transduction techniques were applied to silence and overexpress Ubc9 in MARC-145 cells, respectively. The results indicated that the overexpression of Ubc9 could significantly inhibit PRRSV replication at 24 h pi (P<0.01) and markedly block the genomic RNA synthesis of PRRSV at 24 h piand 36 h pi (P<0.001). In contrast, SUMOylation inhibitor GA was found to be able to enhance the viral replication in MARC-145 cells at 12 h pi(P<0.01) and 24 h pi(<0.001) compared with the control group.Further more, to elucidate the SUMOylation pathway of PRRSV N protein, site-directed mutagenesis technique was adopted to substitute the lysines in N protein. The results showed that SUMOylation was absent only when all lysines were mutated into arginines, but not in single or multiple lysine mutations, which indicated thatthe lysines of N protein are redundant for its SUMOyaltion. As a SUMO E3 ligase, PIAS1 (Protein inhibitor of activated STAT1) can promote the SUMOylation of the target protein, which in turn affects the function of the target protein and participates in the transcriptional regulation process. As well, in vitro SUMOylation detection kit and immunoprecipitation technique were used to identify the SUMOylation pathway of N protein, to verify the interaction of PIAS1 with N protein, and to analyze the role of PIAS1 in SUMOylation of N protein and PRRSV replication. The results showed that PIAS1 could interact with the N protein,and both of them predominantly located in the cytoplasm. However, exogenous transfection of PIAS1 did not increase the SUMOylation level of N protein. Even though, the overexpression of PIAS1 could promote PRRSV replication in MARC-145 cells.Considering the fact that the lysines are redundant for the SUMOylation of PRRSV N protein, a series of mutant virus with N protein lysine mutants were rescued to further explore the relationship of lysines in N protein with its subcellular localization, interferon inducing and PRRSV replication.According to the distribution of lysines in the N protein, a series of lysine mutants were constructed by site-directed mutagenesis with pCMV-HA-N as template. Meanwhile, using the highly pathogenic strain JXwn06 infectious cDNA clone (pWSK-JXwn) as the backbone, lysines were mutated to arginines in N protein. Totally six N protein lysine mutated viruses were successfully rescued and designated as RvJXwn, RvJXNK7,28,39,52R, RvJXNmutNLS1, RvJXNmutNLS2,RvJXNmutNLS 1,2 and RvJXNKR, respectively. To characterize and compare the growth prosperities of these mutants with their parental virus RvJXwn, the growth kinetics were depicted in MARC-145 cells or primary PAMs, when the lysines of NLS1, NLS2, NLS1,2 domains in the N protein and the whole lysines were mutated to arginines, the in vitro proliferation efficiency of mutant viruses was significantly lower than that of their parent virus RvJXwn. The subcellular localization of N protein with lysine mutants and mutant virus were observed by laser scanning confocal microscopy. The results showed that NmutNLS2 and wild type N protein were located in the nucleolus and other N protein mutants were distributed in nucleoplasm rather than nucleolus. In addition, all N proteins of mutant viruses and parental virus had been found locating in the cytoplasm and nucleolus. The effect of N protein and its lysine mutants on the promoter activity of IFN-β and IRFs was analyzed by luciferase reporter assay. The findings suggested that NK7,28,39,52R and wild-type N protein could inhibit IFN-β and IRFs promoter activity, while the inhibitory ability of the rest N protein lysine mutants on the IFN-P and IRFs promoter activity was significantly reduced. The ability to induce IFN-β production of mutant rescued viruses in MARC-145 cells and primary PAMs showed that different mutant rescued viruses-infected host cells have significant differences in IFN-β production ability. Higher expression levels of IFN-β from RvJXNmutNLS1 RvJXNmutNLS2,RvJXNmutNLS 1,2 and RvJXNKR were detected, compared with parental virus RvJXwn at 24 h pi and 36 h pi (P<0.001),while no difference was found between RvJXNK7,28,39,52R and RvJXwn(P>0.05).In conclusion,our research confirmed the interaction of PRRSV Nsp1β,Nsp4, Nsp9, Nsp 10 and N protein with the host cell protein Ubc9, and verified that the lysines are redundant for the SUMOylation of PRRSV N protein. By constructing and rescuing a series of N protein lysine mutants,it was confirmed that N protein lysine mutants could affect IFN-β and IRFs promoter activity and alter the subcellular localization of N protein. Besides the lysine residues in 7,28,39,52 sites, other lysine residues of N protein are also responsible for virus replication and IFN-β production in vitro. |
