Molecular Mechanisms Of PRRSV Replication Regulated By The Host Cellular Proteins-p21 And ILF2

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered the most important pathogen threatening worldwide swine industry. In recent years, the nonstructural proteins (nsps) of PRRSV have been shown to play important roles in viral replication, genome transcription and in the modulation of host immune responses. In the present study, we analyzed the degradation of p21 by the nspll of PRRSV and its biological significance, and investigated the interaction of cellular protein ILF2 with viral nsp9 or nsp2 and its effect on PRRSV replication, in an attempt to reveal the roles of nspll, nsp9 and nsp2 during viral replication process, providing scientific evidence for understanding the molecular mechanisms regulating the replication of PRRSV.The change of p21 protein in PRRSV-infected MARC-145 cells was analyzed by Western Blot.The results showed that PRRSV infection could down-regulate the level of p21 protein in MARC-145 cells in a dose-dependent manner. Flow cytometry analysis further showed that PRRSV infection promoted the cells into S phase by down-regulating p21 protein. MARC-145 cells were synchronized to G0/G1, S and G2/M phases using serum starvation, double thymidine blocking and nocodazole,respectively, and then infected with PRRSV, and the virus titers were examined. The results indicated that the cells synchronized to the S phase were conducive to viral replication. Moreover, the knockout of p21 gene in MARC-145 cells using the CRISPR/Cas9 system was beneficial for the viral replication. Next, the plasmid that was expressing each nsp or structural protein of PRRSV was co-transfected into HEK 293FT cells with the p21-expressing plasmid, respectively. Western Blot analysis showed that the nsp11 was the most pronounced protein that down-regulated p21 among the viral proteins. The plasmids that were expressing four mutants of nsp11 with abolished endonuclease activity or deubiquitination enzyme activity were constructed and transfected into HEK 293FT cells together with the p21-expressing plasmid. The results showed that the endonuclease activity of nsp11 was crucial for its ability of down-regulating p21. Moreover, Real-time PCR assay demonstrated that there was no significant change in the mRNA level of p21 after PRRSV infection or nspll transfection. Degradation assay showed that the nsp11 could degrade p21 by proteasome pathway, and ubiquitination analysis further revealed that the nsp11 degraded p21 through an ubiquitin-independent proteasome-dependent pathway. Taken together, these results suggest that PRRSV exploits the endonuclease activity of nsp11 to down-regulate the p21 protein and promotes cells into the S phase,facilitating the viral replication.Fifty-two cellular proteins interacting with the nsp9 of PRRSV were screened by lentiviral expression system combined with immunoprecipitation and mass spectrometry. Among them, the protein ILF2 was selected for further study. The interaction between exogenous ILF2 and nsp9 or nsp2 was first demonstrated in HEK 293FT cells co-transfected with the ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in the MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while different truncations of nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs.Finally, the knockdown of ILF2 favored the replication of PRRSV, while the over-expression of ILF2 impacted the viral replication in MARC-145 cells. These above data indicate that ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV.In summary, our results showed that PRRSV could exploit the endonuclease activity of nsp11 to down-regulate the p21 protein in an ubiquitin-independent, proteasome-dependent manner and promote cells into the S phase, thus facilitating the viral replication. Cellular ILF2 was shown to interact with the nsp9 and nsp2 of PRRSV, and this interaction could inhibit the viral replication.These findings are helpful for us to further understand the roles of nspl 1, nsp9 and nsp2 in PRRSV replication, and provide scientific evidence for elucidating the molecular mechanism regulating the replication of PRRSV.