The Influence Of Host Protein NPM1, EB3 And HSP47 On The Replication Of PEDV

Innate immune response has been recognized as the first defensive line after virus infection, such as cell apoptosis, antiviral cytokines and inflammatory response. Depending on these mechanisms, host may either induce virus clearance by antagonism or establish useful acquired immune response afterwards. However, suppression of inflammatory response and inhibition of target cells apoptosis, gained through virus long-term evolution, may provide many viruses abilities of repaid replication and systemic infection in the host.Coronavirus is regarded as a worldwide pathogen, is susceptible for most types of domestic poultry and mammals as well as human. Coronavirus has caused panic of human beings since the epidemic of SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV) in 2003 and the appearance of MERS-CoV (Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012. From 2010 to 2013, porcine epidemic diarrhea (PED) causes a major economic losses of livestock industry. More and more investigations were conducted in Coronavirus concerning its pathogenic severity and ability of rapid spread. PED caused by porcine epidemic diarrhea virus (PEDV) belongs to family Coronaviridae, subfamily Coronavirinae, genus Alphacoronavirus. PEDV can induce an acute and highly contagious enteric disease characterized by severe diarrhea, dehydration and a high death rate in pigs of all stages of age, with 100% morbidity and mortality 30～80% of newborn piglets, respectively. Therefore, it is crucial and meaningful to carry out the basic research related to replication mechanism of PEDV.Based on the study of subcellular localization of PEDV N protein, we further confirmed the interaction of NPM1 and PEDV N protein using co-immunoprecipitation and GST-pull down essays. The co-localization of NPMl and PEDV N protein in the nucleolus were confirmed by using confocal microscopy. Amino acids 147-294 of N and amino acids 189-294 of NPM1 were found necessarily for the interaction, which has been identified by using GST-pull down essay, results showed that. In addition, K263R mutation of NPMl can promote the interaction of NPMl and PEDV N protein. During investigating the role of NPM1 protein in the subcellular localization of N protein, we observed that nucleolar localization of N protein was independent of the nuclear-cytoplasmic shutting of NPM1 protein. A significant up-regulation of NPMl protein was found in PEDV infected cells. Meanwhile, the NPM1 protein overexpression can promote the expression level of N protein as well as PEDV replication. In contrast, silence of NPMl with small interfering RNAs can inhibit the expression level of N protein and PEDV replication. Subsequently, we found that N protein binding to NPM1 can protect it from proteolytic degradation by caspase-3, then improving of cell survival rates, avoiding premature cell apoptosis and promoting virus replication.Virus-host interaction can be observed at the prophase of virus infection. The interactions between M and EB3 proteins as well as Nsp7 and HSP47 were identified by using yeast two-hybrid assays and GST-pull down assays, respectively. Furthermore, co-immunoprecipitation and confocal microscopy were used to confirm the interaction of M-EB3 and NSP7-HSP47. Intriguingly, single and co-expression of NSP7 and HSP47 showed different outcomes in this study, NSP7 was only found in nucleus and HSP47 in cytoplasm during single expression of each protein. In contrast, we observed both NSP7 and HSP47 localized in perinucleus when they were co-expressed. These observations indicated that the interaction between these two proteins altered subcellular distribution. Another vital role of this study is whether M-EB3 or NSP7-HSP47 interaction can influence the replication of PEDV. Our results showed the polarized influence of M-EB3 and NSP7-HSP47 interaction on the replication of PEDV. The expression level of EB3 protein was significantly up-regulated in PEDV infected cells. The endogenous EB3 protein expression level was increased after transfecting exogenously expression plasmid, and then Vero E6 cells were infected by PDEV at 12 h post-transfection. The results demonstrated that it can induce higher expression level of M protein and a significant increase of PEDV replication. In contrast, the interaction of NSP7-HSP47 can hinder PEDV replication. Our results showed that HSP47 protein was significantly decreased at 48h and 60h post-infection. Increased endogenous HSP47 protein by transfection of recombinant plasmid into Vero E6 can significantly inhibit N protein expression and PEDV replication. The molecular mechanism, how the M-EB3 and NSP7-HSP47 interactions regulate the replication of PEDV, remains to be further elucidated in the furture.