| Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS), is commonly known as blue-ear disease. This disease may infect pigs at all ages, causing breathing difficulties, reproductive disorders and piglet death symptoms. The causative agent of reproductive and respiratory syndrome is reproductive and respiratory syndrome virus (PRRSV), which was first isolated in1991in the Netherlands, and were then isolated in1996in China. PRRSV is an enveloped single strand RNA viruses belonging to the family Arteriviridae. Virus particles are spherical with a diameter about55to60nm in length. Currently, the mechanisms of replication and molecular pathogenesis of PRRSV remains largely unknown.Ubiquitin is a small protein (8KDa) widespread in vivo, consisting of76amino acid residues. The sequence of ubiquitin is highly conserved among different species. Previous studies demonstrated that ubiquitin-proteasome system plays a very important role in the degradation of endogenous protein and regulation of many cellular functions. Furthermore, ubiquitin-proteasome system also plays a vital role at different stages of viral infections. It is evident that various viruses utilize and regulate the ubiquitin-proteasome system in target cells for their replication. Interest has therefore shifted to questions of whether ubiquitin-proteasome system participates in the course of PRRSV infection. In this study, we investigate the roles of the ubiquitin-proteasome system in the process of PRRSV infection. The main contents are as follows:1. In the process of marc-145cells infected with PRRSV, virus manipulate the degradation of intracellular ubiquitinated-proteinMarc-145cells were inoculated with PRRSV or PBS to investigate the relative changes between intracellular free ubiquitin and poly-ubiquitination. The samples, harvested at different times after infection, were analyzed by Western Blotting. The results show that during24-42h after virus infection, the expression of protein ubiquitination was drastically reduced, and free ubiquitin was increased. After42h, the expression of protein ubiquitination has returned to the original level since virus infection. In the negative control group, a slight change in expression level of protein ubiquitination was found throughout the treatment. The results demonstrated that the degradation of ubiquitinated-protein is prerequisite for successful reproductin of PRRSV during the infection.2. Inhibition of the26S proteasome function is not conducive to PRRSV proliferationThree proteasome inhibitors were used to treat Marc-145cells before PRRSV inoculation. Samples were harvested at24h. The virus titer was determined previously by plaque assay. We noticed that two inhibitors of three significantly can inhibit viral proliferation. Marc-145cells were treated with different concentrations of inhibitors; samples were harvested at24h after PRRSV inoculation; the changes of viral titer were determined by plaque assay. The results showed that the virus titer was dose-dependent decrease. Furthermore, we used MG132(2μM) and bortezomib (1μM) to treat cells; the samples were harvested at different times after PRRSV inoculation, the changes of viral titer was determined by plaque method. Interestingly, the tendency of virus reproduction is alike between inhibitors treated groups and negative control group, but titers of inhibitors treated groups collected at different time points were all reduced accordingly.3. Proteasome inhibitor affects the production of PRRSV Nsp2proteinMarc-145cells were treated by using different concentrations of inhibitor treatment. Samples were harvest24h after PRRSV inoculation and the expression of the PRRSV Nsp2protein was detected by Western Blotting assay. The results show that with the increase in the dose of inhibitors, the virus protein expression was reduced gradient. Marc-145cells were treated with MG132(2μM) and bortezomib (1μM); samples were harvested at different times after PRRSV inoculation; the changes of the expression PRRSV Nsp2protein were determined by Western Blotting assay. The results demonstrated that the inhibitors delay and hamper the expression of PRRSV Nsp2protein.4. Proteasome inhibitor affects middle stages of PRRSV infectionMarc-145cells were inoculated with PRRSV, and proteasome inhibitors MG132(2μM) and bortezomib (1μM) were used to treat samples at different times. The effectiveness of proteasome inhibitors activity was confirmed by the analysis of plaque assay. Notably, following proteasome inhibitors, production of PRRSV was heavily decreased, particularly in16-24h after inhibitor treatment. 5. Over expression of ubiquitin can enhances PRRSV proliferationMarc-145cells were transiently transfected with the ubiquitin eukaryotic expression plasmid pCMV-(HA-Ub)4, then infected with PRRSV, the over-express groups and negative control groups were treated with proteasome inhibitors or DMSO24h later respectively. Cells were harvest at24h and the virus titer was detected by plaque assay. Notably, we noticed that treating with proteasome inhibitors cause accumulation of polyubiquintined protein in cytoplasm, and viral titers were significantly increased, suggesting that the over expression of ubiquitin can enhances PRRSV proliferation. |
