Replication Of Classical Swine Fever Virus And Autophagy

Classical swine fever(CSF) is a pig’s viral diseases caused by Classical swine fever virus(CSFV). The typical symptoms and pathological features of CSF are high fever and the organization hemorrhage. CSF cause serious economic losses and challenges to China’s pig industry due to its high infection and fatality. CSFV is a single-stranded positive RNA virus, classified into the pestivims genus, Flaviviridea family. As the other members of the pestivims genus, CSFV proliferates rapidly and causes lethal damage in vivo, but showes a persistent infection and does not cause the cytopathic effect(CPE) in cultured cells. Virus, as the strict cells parasitic organisms, are multiplied and replicated based on host resistance and virus evading strategy, even controlling of host cell physiological mechanism. So, it is necessary to investigate the relationship between CSFV and cell physiological activities, this is helpful for us to understand the CSFV pathogenesis.Autophagy is an evolutionally conserved cellular physiological mechanism in eukaryotes and important for the maintenance of cellular homeostasis. Autophagy is induced rapidly in eukaryotic cell when cells met some stress factors such as starvation, lack of growth factors, energy requirement or invasion of pathogenic microorganisms. Autophagy phagocytosed and degras the cellular aging organelles, long-lived proteins and exogenous pathogenic microorganisms by fused with lysosome. The degradation products are recycled to maintain the function of cellular organelles and cellular homeostasis. In addition, autophagy has the important function to defense bacteria and viruses infections. However, recent reports reported that many viruses were not controlled by cell autophagy mechanism but manipulated autophagy mechanism to serve for their proliferation. In this study, we investigated the relationship between CSFV and cellular autophagy, the results as follow:(1)The impact of CSFV on the cellular autophagy. CSFV could induced the formation of the double-membrane of autophagosome and GFP-LC3 dot in ST cells, and up-regulated the expression of autophagy associated gene Beclin-1 and LC3 when CSFV Shimen strain infected the ST cells, detected by transmission electron microscope(TEM), MDC staining, LC3 fluorescence and Western blot. These results suggested that CSFV infection could induce autophagy in ST cells. CSFV promoted the degradation of autolysosome substrate protein p62 after infection, but increased the expression of p62 when CQ blocked the formation of autolysosome, this suggested that CSFV infection could induce the formation of autolysosome and promote the autophagy process. UV inactivated virus could induce autophagy at 0.5h post infection not the latter infection phase, this implied that autophagy induction requied virus replication, as well as the adsorption to the cell membrane could also induce autophagy. These results showed that CSFV could induce autophagy and promote the autophagy process in ST cells.(2)The affection of the CSFV structural and no-structural proteins on autophagy mechanism. The eukaryotic expression vector of CSFV structural and no-structural proteins were structured for evaluating the affection on autophagy in ST cells, differential expressions of autophagy associated genes by quantitative real time PCR and Western blot. It was found that the structural protein E1 and no-structural protein NS4 B could up-regulate the expression of autophagy associated gene Beclin-1 and LC3 and promote the degradation of p62 protein, the no-structural protein NS2 and NS3 could up-regulate the expression of Beclin-1 and LC3 but had no influence on the expression of p62. These suggested that E1, NS4 B induced the autophagy and promoted the complete autophagy process, and the NS2 and NS3 only induced the autophagy but not the complete autophagy process.(3)The influence of autophagy on the CSFV replication in ST cells. The influences of autophagy on the CSFV replication were evaluated with autophagy promoter rapamycin, inhibitor 3MA and autophagy flux blocker CQ. The results showed that autophagy promoter rapamycin and autophagy flux blocker CQ both promoted the CSFV replication, the inhibitor 3MA inhibited viral replication. There was no significant impact on the CSFV replication when autophagy regulators were added in ST cells after CSFV infection. Furthermore, it was confirmed that the suppression of autophagy process was adverse for CSFV replication by RNAi of autophagy associated genes. In conclusion, the autophagy process, especially the formation of autophagosome could promote the CSFV replication in ST cells.(4)The influence of CSFV induced autophagy on the cell cycle and proliferation of ST cells. The influence of CSFV induced autophagy on the cell cycle and proliferation of ST cells were detected by CCK-8 Kit, Flow cytometry, q RT-PCR of cyclins and Brd U staining, it was found that the number of cells in S-phase and DNA synthesis increased, but the cell proliferation was not promoted when infection. In autophagy associated genes(Beclin1 and Atg7) mute ST cells, CSFV infection could suppress cell proliferation, prevent DNA synthesis, and arrested ST cells in G0/G1 phase. These suggested that autophagy involved in the regulation of CSFV infection on the cell cycle of ST cells.To sum up, this study illustrated: CSFV infection induced the autophagy in ST cells and promoted the complete autophagy process;the E1 and NS4 B protein of CSFV could induce complete autophagy process, the NS2 and NS3 only started the autophagy but not induced the complete autophagy flux; the autophagy process benefited the CSFV replication in ST cells;autophagy involved in the regulation of CSFV on the cell cycle and replication of ST cells.