| Pseudorabies virus(PRV) is the causative agent of Aujeszky’s disease, producing reproductive failure in sows, respiratory disorders in fattening pigs, and fatal encephalitis in piglets, and resulting in heavy losses to the swine industry. Owing to the property of latent infection, it is hard to clear PRV in swine industry. IL-10 is a pleiotropic and important cytokine. Recently, it has been reported that IL-10 protected cells latently infected with virus from death, contributing to store viral genome after a latent infection.As a consequence of translation arrest, stress granules(SGs) play a pivotal role in protecting cells under conditions of stress, including heat shock, starvation, oxidation, ultraviolet radiation, hypoxia, and viral infection. However, some viruses could induce the formation o f SGs, whereas others actively inhibit their formation.In this study,we designed experiments to investigate the influence of PRV infection on IL-10 and SGs. The main contents were as follows:1. PRV upregulated IL-10 at promoter, mRNA, and secretionLuciferase reporter assays, Real-time RT-PCR and ELISA were performed to detect the IL-10 promoter, m RNA, and the secreted IL-10, respectively. IL-10 promoter was significantly upregulated at 6 hpi, and PRV induced IL-10 promoter in a time-dependent and dose-independent pattern. However, PRV induced IL-10 m RNA from 12 hpi in a time and dose dependent pattern. Further more, PAMs were infected or mock-infected with PRV, we found that PRV induced secreted IL-10 from 12 hpi. UV-inactivated PRV failed to induce IL-10 at promoter, m RNA, or protein levels, indicating that upregulating IL-10 closely related to PRV replication.2. Hypomethylation of IL-10 promoter participated in upregulating IL-10 upon PRV infectionBSP and transfected with plasmids encoded po DNMTs were performed to analyse the methylation of IL-10 promoter and the influence of po DNMTs on IL-10 upon PRV infection. In PK-15 cells, we found hypomethylation of IL-10 promoter upon PRV infection. Overexpression of DNMT3 a or DNMT3 b could restrict the effect of PRV inducing IL-10, demonstrating that Hypomethylation of IL-10 promoter participates in upregulating IL-10 upon PRV infection.3. PRV infection did not induce SG accumulationTo investigate whether PRV infection induces SG accumulation, He La cells were mock-infected or infected with PRV at an MOI of 1. The presence of SGs was assessed with IFA at several time points after infection with an antibody specifically directed against the SG marker TIA-1. The structural protein g B encoded by PRV was also detected with IFA to determine the progression of infection. There are no SGs accumulated during PRV infection, at all the time points and doses examined. To determine whether the lack of SG accumulation during PRV infection was cell type dependent, we performed similar analyses in PK-15 cells, and did not observe SG accumulation in PRV-infected PK-15 cells. Taken together, these results indicate that no SGs accumulated during PRV infection.4. PRV infection suppressed the SG formation induced by Arsenite and HippuristanolIFA was performed to analyse the influence of PRV infection on SG accumulation upon treated with Arsenite or Hippuristanol. PRV infection suppressed the SG formation induced by a classical SG inducer(arsenite) and a nonclassical SG inducer(hippuristanol). This inhibition appeared to be independent of e IF2? phosphorylation. Western-blotting analysis with specific antibodies directed against TIA-1 was performed to investigate whether PRV infection influences the constituent proteins of SGs, we found PRV infection caused a slight reduction in the levels of TIA-1 from 9 hpi, and TIA-1 was decreased more in the groups with PRV infection and arsenite treatment from 6 hpi... |
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