NS5A Protein Of Classical Swine Fever Virus Interactes With Host Cells HSP70 And Induce The Unfolded Protein Response

Classical swine fever(CSF),as a violent and contagious disease in pig, is caused by classical swine fever virus(CSFV) and characterised by a high fever, generalized bleeding and immunosuppression. The disease is spread quickly, with a high death rate, different ages and breeds of pigs can be infected, is one of the must be reported zoonotic by World Animal Health(OIE) and also listed as one class of animal disease in our country. CSFV is a enveloped virus, with a single-strand, positive-sense RNA genome, which is belong to Flaviviridae Pestiviruses member. NS5 A protein is one of the CSFV non-structural protein, is also an important part of the viral replication complex. In this study, we centre on CSFV NS5 A protein, research its interaction with the host cell HSP70 protein and how this interaction affects the replication of the virus itself, as well as how NS5 A protein activates the host cell unfolded protein response(UPR) and involved the signal pathway. Around this research, we obtained some results showed as the following:1. CSFV NS5 A protein interact with the host HSP70 protein. At the protein level, we are utilizing CO-IP technology and GST-Pulldown in intracellular and extracellular verify NS5 A and HSP70 protein were capable of binding to each other respectively, and verifyed that the N terminal of NS5 A protein is responsible for the interaction with HSP70 by CO-IP method; at the cellular level, we proved that NS5A/HSP70 proteins colocalization in the cell by confocal laser technology.2. HSP70 protein can promote CSFV gene replication. NS5 A protein is involved in replication of viral genes since it is a member of the viral replication complex of CSFV. NS5 A protein has been shown the existence of interactions with HSP70, we used different methods to achieve HSP70 overexpression in cells, and sh RNA-mediated lentiviral silence of HSP70 gene or use specific inhibitors of HSP70 proteins quercetin reducing the protein level of HSP70, the results both shown some positive correlation between HSP70 expression level and the amount of CSFV gene replication in the cells. That instructions that HSP70 protein plays a significant role in promoting the gene replication of CSFV. Interestingly, we found that in ST cells infected with CSFV, the expression of HSP70 protein level also showed a clear upward trend. That indicate CSFV itself can increase the expression of HSP70, with a result this could have advantage in proliferation of the virus itself.3. CSFV infection or exogenous expression NS5 A protein in ST cells can induce cells UPR, and the activation of UPR signaling pathways are consistent, both activate ATF6 and IRE1 pathways, less impact on the PERK pathway. ATF6 and IRE1 signaling pathways in UPR activated help the unfolded or misfolded protein ro refolding or degration, aim to remission the endoplasmic reticulum stress, maintain homostasis of the cell; the PERK parthway activated in the later of stage, the mechanism of apoptosis of cells will be activated, finally can achieve the effect of inducing cell apoptosis. We used q RT-PCR and Western blotting technique to study the gene and protein expression level of key protein whithin UPR signaling pathways in cells when infected with CSFV or exogenous expression NS5 A protein. And confirmed both of them induced UPR through activation of ATF6 and IRE1 pathway.4. Use the method of point mutations studies confirmed 81 th serine and 401 th threonine in NS5 A protein are key amino acid sites in the process of activation UPR pathway. Compared to normal NS5 A protein, when its 81 th serine and 401 th threonine was replaced by alanine, the effect of the ability of NS5 A protein upregulation of ATF6 and GRP78 expression level was disappear.In summary, CSFV NS5 A protein not only can through the interacton with host cell HSP70 play a positive role in the replication of the virus itself, but also can activate the host cell UPR, to inhibit apoptosis cells, to enhance the capacity of cellular protein synthesis, to better service for viral proliferation. The results obtained in this study, offer a new knowledge of fully understand the physiological function of CSFV NS5 A protein, and also provides some new theoretical data to understand the deep relationship between the virus and the host cells.