Function And Mechanism Of Non-Muscle Myosin ⅡA(NMⅡA)Mediated PEDV Infection

Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV),is an acute,highly contagious swine intestinal infectious disease,which is characterized by a series of symptoms including vomiting,diarrhea and dehydration.PEDV infects pigs of different ages and often causes high morbidity and mortality in piglets,thus seriously threatening the healthy development of pig industry and leading to huge economic losses in all countries worldwide.Therefore,in-depth investigation of host factors in target cells and their interactions with PEDV is the basis for revealing the molecular mechanisms of infection and pathogenicity of this virus.In mammals,there are three non-muscle myosin Ⅱ(NM-Ⅱ)isoforms or paralogs,which are composed of a pair of identical essential light chains(ELC)and a pair of regulatory light chains(RLC),and a pair of different heavy chains(i.e.,NMHC-ⅡA,B and C,encoded by MYH9,MYH10,and MYH14 genes,respectively),correspondingly,referred to as NM-ⅡA,ⅡB,ⅡC.These proteins are widely distributed throughout the organism and have important biological functions in cell adsorption,migration,cytokinesis,etc.Meanwhile,increasing evidence indicates that NM-Ⅱ,especially NM-ⅡA,also plays an important role in the process of infection of various viruses,such as herpes simplex virus type 1(HSV-1)and Epstein Barr virus(EBV).For instance,these viruses are regarded to utilized NM-ⅡA as a cell receptor to achieve successful infection.In this study,the expression level of NM-ⅡA was found to be significantly up-regulated after PEDV infection both in vivo and in vitro,suggesting that the alteration of NM-ⅡA expression might be associated with the infection of this virus.Subsequent investigations indeed confirmed that NM-ⅡA promoted the infection of PEDV to target cells,this conclusion was derived from the following analyses.1.Analysis of the interplay of NM-ⅡA with PEDV infectionAt the tissue level,the co-localization of NM-ⅡA and PEDV on small intestinal villi was found in PEDV-infected small intestine tissues by indirect immunofluorescence assay.Moreover,the m RNA and protein levels of NM-ⅡA in PEDV infected small intestine tissues were significantly higher than those of the control.Similar phenomena were also observed in PEDV infected Vero cells,and PEDV and NM-ⅡA were mainly co-localized near the cell membrane.On the contrary,the inhibition of NM-ⅡA ATPase activity by using its inhibitor led to pronouncedly compromised PEDV replication,as demonstrated by a significant decrease in viral copy number.These results indicate that the function of NM-ⅡA and PEDV infection have a mutual influence.2.The effects of overexpressed full-length molecule and functional domain or downregulated NM-ⅡA on the PEDV infectionTo further analyze the function of NM-ⅡA in PEDV infection,full-length NM-ⅡA overexpression plasmid and shRNA-mediated targeting monkey NM-ⅡA gene plasmid were constructed,and corresponding empty plasmid was prepared as negative control.After antibiotic screening and monoclonal selection,Vero cell lines stably overexpressing and interfering NM-ⅡA were obtained for subsequent experiments.The results showed that overexpression of NM-ⅡA gene significantly promoted PEDV infection,while knockdown of this gene presented completely opposite consequence.Subsequently,plasmids containing the head or tail functional domain of NM-ⅡA protein were constructed and transfected into Vero cells,respectively.Intriguingly,the results indicated that overexpression of the NM-ⅡA tail domain considerably promoted PEDV infection,while the head domain had no obvious effect,suggesting that the functional domains of NM-ⅡA have different influence on PEDV infection.3.Investigations on the interactions between NM-ⅡA and PEDV virus structural proteinsIn order to determine NM-ⅡA interacts with which structural proteins of PEDV to achieve its functions during PEDV infection,four eukaryotic plasmids individually expressing PEDV structural proteins(S,N,M and E)fused with different tags were constructed,and then transfected into 293 FT cells,respectively.The results demonstrated that the expression of NM-ⅡA was significantly up-regulated only in cells transfected with PEDV-S or PEDV-E expression plasmid,with the former more pronounced.Moreover,a co-localization of PEDV-S with NM-ⅡA was observed.Importantly,the interaction between the two proteins was further confirmed by coimmunoprecipitation assay.In summary,this study confirmed that NM-ⅡA displays a promoting action on PEDV infection,and this effect seems to be closely related to the tail functional domain of this protein,moreover,its interaction with the PEDV S structural protein may facilitate the achievement of this function.Therefore,these new findings expand the spectrum of viruses affected by the function of NM-ⅡA,lay a foundation for further investigations on the functions of PEDV structural proteins S and E in mediating the role of NM-ⅡA in different stages of PEDV infection,such as adsorption,internalization,assembly release,etc.,as well as provide new insights into the PEDV pathogenesis,novel target for the prevention and treatment of PED,and scientific basis for improving the efficacy of vaccines.