The Role Of IL-1β And Cholesterol In Classical Swine Fever Virus Infection

Classical swine fever(CSF), caused by classical swine fever virus(CSFV), is a highly contagious viral disease affecting pigs and is designated a notifiable disease by the Office International des Epizooties. CSFV has a tropism for vascular endothelial cells and immune system cells. Cholesterol-rich lipid rafts have been proved to be involved in various steps of the life cycle of many enveloped and even nonenveloped viruses. In this study, we investigated the production of IL-1β from macrophages following CSFV infection and the role of cholesterol in CSFV infection. The results were shown as follow:(1) Using Real-time PCR and ELISA, our results showed that IL-1β was upregulated after CSFV infection. Our results also showed that caspase-1 was activated by CSFV infection through western blot, which implied that CSFV-mediated IL-1β release through activating caspase-1. Subsequent studies demonstrated that CSFV trigger reactive oxygen species(ROS) accumulation. Using ROS inhibitor pyrrolidine dithiocarbamate(PDTC), we demonstrated that ROS may not be involved in CSFVmediated IL-1β release. These results demonstrated that CSFV infection could induce the secretion of mature IL-1β through activating caspase-1, whilst ROS was not involved in this process.(2) For amplification of the CSFV p7 gene, the PCR oligonucleotides were designed according to the archived CSFV Shimen strain nucleotide sequence. The PCR product was subcloned into the mammalian expression vector p EGFP-C3. The recombinant plasmid was sequenced and named p EGFP-p7. Using Lipofectamine 2000 reagent, the plasmids were transfected into SUVECs and macrophages. By adding 1500 μg/ml G418, we obtained stably transfected cell lines expressing fusion proteins GFP-p7. Recently, researches have indicated a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induced IL-1β secretion which could be inhibited by the ion channel blocker amantadine and also discovered that p7 protein was a short-lived protein in different cell types degraded by the proteasome.(3) For depletion of cellular cholesterol, macrophages were seeded in 12 wells plate and treated with different concentrations of Methyl-β-cyclodextrin(MβCD) at 37 ℃ for 1h followed by CSFV infection. The results showed that depletion of cellular cholesterol affects CSFV entry in a dose-dependent manner. We further examined the effect of cellular cholesterol on post-entry stage of CSFV infection; the results demonstrated that cellular cholesterol plays a critical role in the intracellular replication steps of CSFV. For cholesterol replenishment, after extraction of cellular cholesterol, exogenous cholesterol was added in for another 1 h. The trans-supplementation experiment demonstrated that replenishment of cholesterol by adding exogenous cholesterol partially restored CSFV infectivity with the increase of viral m RNA both in the viral entry and post-entry stage.(4) For depletion of viral envelope cholesterol, CSFV was mixed with MβCD at 37 ℃ for 1h. For cholesterol replenishment, exogenous cholesterol at a concentration of 200 μg/m L was added, and then the mixture of virus, MβCD and cholesterol was incubated for another 1 h at 37 ℃. Then, to avoid the effect of drugs on cells, the samples were diluted in 1640 without serum at least 100-fold. CSFV was found to be sensitive to MβCD with a significantly reduction of viral RNA and also virus infection was partially recovered when exogenous cholesterol was added to replenish the envelopes of MβCD-treated CSFV virions. These data demonstrated that cholesterol in the viral envelope plays an important role in the infectivity of CSFV.(5) To establish CSFV’s influence on Caveolin-1expression, macrophages were infected with CSFV. At specified time points, total cellular RNA and protein were extracted from mock infected or CSFV infected cells and the expression of Caveolin-1 was quantified by real-time reverse transcription(RT)-PCR or Western blot analysis. The data suggested that CSFV infection enhance Cav-1 expression. Caveolin-1 knock-down experiments showed that caveolin-dependent endocytosis may not play a role in CSFV infection.In summary, the results showed that CSFV infection induced the expression and production of IL-1β through activating caspase-1, whlist ROS was not involved in this process. The data also demonstrated that CSFV viroporin p7 protein induced IL-1β secretion that could be inhibited by the ion channel blocker amantadine, which provided some new insights in CSFV-mediated inflammatory processes. In addition, our results showed that depletion of cellular cholesterol affects CSFV entry and also it plays a critical role in the intracellular replication steps of CSFV, which can be partially reversed by replenishment of cholesterol. Subsequent studies demonstrated that CSFV infection up-regulated caveolin-1expression, while caveolin-dependent endocytosis seems not to be involved in CSFV infection by performing Cav-1 knock-down experiments. These data will provide some evidence on role of lipid raft in CSFV infection.