| PRRS is one viral infectious disease severely affecting the swine industry worldwide. Its causative agent is PRRSV. In recent years, the biological function of PRRSV Nsp on pathopoiesia such as virus replication and immunosuppression, and the interaction with host cell protein have been becoming an hot area for PRRSV study. In our study, we deeply investigated the interaction of PRRSV Nsp9with pRb and its molecular mechanism in order to show the function of Nsp9on replication and cell cycle modulation, and provide scientific basis for pathogenic mechanism of PRRSV.The amino acid sequence of the Nsp9among17representative strains of PRRSV was aligned. The results indicated that the Nsp9of all the analyzed PRRSV shared an identical LXCXE motif (LACAE). A1914bp cDNA fragment of truncated porcine pRb gene (pRb-tru) including the LXCXE motif was amplified by RT-PCR and plasmid pCMV-Myc-pRb-tru expressing pRb-tru was constructed; meanwhile, plasmids pCMV-HA-Nsp9expressing Nsp9and [pCMV-HA-Nsp9(L415A/C417A), pCMV-HA-Nsp9(E419K)] expressing LXCXE mutational Nsp9were constructed. HEK293T cells were co-transfected with pCMV-Myc-pRb-tru and pCMV-HA-Nsp9, pCMV-Myc-pRb-tru and pCMV-HA-Nsp9(L415A/C417A), pCMV-Myc-pRb-tru and pCMV-HA-Nsp9(E419K), and the co-immunoprecipitation assay was performed. The results showed that Nsp9could interact with pRb through the LACAE motif. MARC-145cells were co-transfected with pCMV-Myc-pRb-tru and pCMV-HA-Nsp9, meanwhile, MARC-145cells and PAMs were infected with PRRSV. The laser scanning confocal microscope assay was performed and the results indicated that not only in transfected MARC-145cells, but also in PRRSV infected MARC-145cells and PAMs, Nsp9and pRb-tru (or endogenous pRb) could co-localize in the cytoplasm. MARC-145cells were transduced lentivirus expressing pRb or transfected pRb siRNA, and the effect of over-expressing and disturbing pRb on PRRSV replication was analyzed. The results showed that the virus titers in the over-expressing pRb-tru groups significantly reduced compared with the control groups (p<0.001), indicating that the over-expression of pRb-tru could inhibit the replication of PRRSV; the virus titers of sipRb groups were significantly higher than the siCon group (p<0.001or p<0.01), indicating that silencing pRb gene facilitated the replication of PRRSV. These results suggested that host cell protein pRb could inhibit the replication of PRRSV. Western blotting analysis indicated that PRRSV infection down-regulated the level of pRb. The results of degradation and ubiquitination assay showed that Nsp9could promote the degradation and ubiquitination of host cell protein pRb. These results suggested that Nsp9down-regulated pRb by ubiquitin proteasome pathway and antagonized the inhibition function of pRb on PRRSV, facilitating PRRSV replication.To further examine the molecular mechanism of Nsp9degrading pRb. Co-immunoprecipitation assay was performed and the results indicated that E3ubiquitin ligase E6AP could interact with Nsp9and pRb. The laser scanning confocal microscope assay indicated that both Nsp9and E6AP, pRb and E6AP could co-localize in the cytoplasm not only in MARC-145cells over-expressing Nsp9and E6AP, but also in PRRSV infected MARC-145cells and PAMs. The results of degradation and ubiquitination assay showed that Nsp9promoted E6AP dependent degradation and ubiquitination of pRb. Co-immunoprecipitation assay was performed to confirm which domain of Nsp9interacted wih pRb and the results indicated that the N terminal domain of Nsp9could interact with E6AP. These results suggested that the RdRp domain of Nsp9could recruit the pRb to E6AP, promoting E6AP dependent degradation of pRb in the cytoplasm by ubiquitin proteasome pathway.The effect of the interaction of Nsp9with pRb on the cell function was analyzed. Luciferase reporter assay was performed to detect the effect of Nsp9on E2F responsive promoter p107. The results showed that Nsp9could specifically activate the E2F responsive promoter p107. The results of Western blotting showed that Nsp9up-regulated the level of p107and the results of flow cytometry indicated that the interaction of Nsp9with pRb increased the S phase entry of cell cycle. These results suggested that the interaction of Nsp9with pRb specifically activated the E2F responsive promoter p107and modulated the cell cycle progression.To further examine the effect of cell cycle on PRRSV replication. MARC-145cells were treated with aphidicolin and DMSO to make cells arresting at G1and S phase and the virus titers were measured. The results showed that the increase of S phase facilitated PRRSV replication (p<0.001).In conclution, our date showed that the Nsp9recruited pRb to E6AP, promoted E6AP dependent degradation of the pRb by ubiquitin proteasome pathway, activated the E2F responsive promoter p107and promoted the S phase entry of cell cycle, facilitating PRRSV replication.Our study provided scientific basis for understanding the function of Nsp9on PRRSV replication and pathogenic mechanism. |
