The Effects Of Type ? Transmembrane Serine Proteases (TTSPs) On The Spike Protein Of Porcine Epidemic Diarrhea Virus And Viral Replication

Porcine epidemic diarrhea(PED)is caused by porcine epidemic diarrhea virus(PEDV),which is an acute and highly contagious enteric viral disease in nursing pigs.PED is characterized by vomiting and lethal,watery diarrhea,which has become one of the most important diseases affecting piglets death.The outbreak and epidemic of PED has brought tremendous economic losses to the pig industries.Recently,the effectiveness of the PEDV vaccine has been questioned because PED outbreaks have also occurred in vaccinated herds,which might be due to the variation of PEDV epidemic strains.Therefore,exploiting the mechanisms of PEDV infection,establishing effective methods of PEDV isolation,developing novel drugs and vaccines has been a problem that many researchers paid close attention to.However,the difficulty of PEDV isolation and low viral titers in the culture brings obstacle to the further study of PEDV in practice.Recently,a type of trypsin-like serine protease,termed type ? transmembrane serine protease(TTSP),was reported to cleave and activate influenza virus and coronavirus surface proteins,allowing multicycle replication in the absence of trypsin.However,the effects of transmembrane serine proteases on host infection by animal coronaviruses,especially PEDV,have not been thoroughly studied thus far.In this study,TTSPs including transmembrane protease serine 2(TMPRSS2),human airway trypsin-like protease(HAT),differentially expressed squamous cell carcinoma gene 1(DESC1),and mosaic serine protease long-form(MSPL)were tested for their ability to facilitate PEDV replication in Vero cells,and evaluated the effects of TTSPs on the spike(S)protein of PEDV.PEDV may be trypsin-independent after serial passages.We firstly confirmed the trypsin dependence of the high and low generation of PEDV LJB/03(P23 and P146)in our lab.Through the viral CPE,immunofluorescence and the S protein sequences analysis,we found LJB/03 P23 was trypsin-dependent.However,the virus culture of LJB/03 P146 in the present or absent of trypsin was not different significantly,which demonstrated that LJB/03 P146 has been incompletely trypsin-dependent.The sequences analysis of S protein showed that the mutations of LJB/03 P146 were the same as the other attenuated strains,which suggested that LJB/03 P146 strain may tend to attenuation.To exploit the effects of TTSPs on PEDV culture,PEDV LJB/03 P23 and P146 were selected to analysis the effects of TMPRSS2,HAT,DESC1 and MSPL on the trypsin-dependent and incompletely trypsin-dependent PEDV replication in Vero cells.The results showed that TMPRSS2 and MSPL could facilitate the trypsin-independent PEDV replication,and promote the viral titers in Vero cells.MSPL displayed predominant promotion effects better than 3 ?g/m L trypsin treatment,but not HAT and DESC1.PEDV LJB/03 P146 was not sensitive to TTSPs and trypsin.To assess whether TTSPs could facilitate the isolation of PEDV in vitro,we processed the PEDV-possitive small intestine tissues and cultured in Vero cells expressing TMPRSS2,HAT,DESC1 and MSPL.We confirmed that TMPRSS2 and MSPL can effectively facilitate the isolation of PEDV in vitro in the absence of trypsin.Viral adaptation and growth in Vero cells expressing TMPRSS2 and MSPL were higher than those in Vero cells expressing HAT and DESC1 as well as trypsin treatment.Preliminary study on the promoting effect of TTSPs on PEDV in Vero cells showed that TMPRSS2 and MSPL could induce membrane fusion of cells infected by PEDV using the cell-cell fusion assay.MSPL could promote cell fusion better than 1 ?g/mL trypsin treatment,but TTSPs did not active LJB/03 P146 for cell-cell fusion.To evaluate the effects of TTSPs on PEDV entry and replication,the TTSP inhibitor was used in this study.The viral RNA levels were decreased in a dose-dependent manner following TTSPs inhibitor AEBSF-HCl treatment,which demonstrated that the TTSPs inhibitor might effectively inhibit the entry of virions.Immunofluorescence staining of TTSP-transfected Vero cells infected with PEDV revealed that the PEDV was extensively co-localized with MSPL and TMPRSS2 but not with HAT or DESC1.To exploit the mechanism of TMPRSS2 and MSPL facilitating PEDV replication in Vero cells deeply,the parent and P146 S gene plasmids were constructed for analysing the effects of TTSPs on S protein of trypsin-dependent and incompletely trypsin-dependent PEDV strains.The result showed that trypsin could cleave S protein with 150 k Da cleavage fragment,which suggested that the 890 amino acid in S2 domain was the cleavage site of trypsin.This cleavage may induce viruscell fusion and promote viral entry.To analysis the cleavage of TTSPs on S protein of PEDV,the TTSPs and S recombinant plasmids were co-transfected in Vero cells.The result suggested that TMPRSS2 and MSPL could cleave S protein with 35 k Da cleavage fragment,but not HAT and DESC1.The cleavage site might be in the S1 domain which have the protein and sugar receptor binding regions.S protein cleaved by TMPRSS2 and MSPL may expose the receptor binding region,which facilitates virions to combine with cell receptors and enhances PEDV infection.The co-localization of the four TTSPs with the PEDV S protein was investigated in infected Vero cells to determine the mechanism of PEDV activation by TTSPs.With the PEDV pseudoviruse packed by S protein,we further confirmed that TMPRSS2 and MSPL could active S protein for virus entry,which may be related to the activation by cleaved S protein.Through the cell-cell assay,we found TMPRSS2 and MSPL could active S0 and S146 protein for facilitating cell-cell fusion.Moreover,we confirmed the presence of TMPRSS2,HAT,DESC1,and MSPL in the small intestines of normal piglets,and we also found that the m RNA levels of these TTSPs increased in PEDVinfected small intestine tissues,which may be mediated by PEDV for facilitating viral replication and spread.In conclusion,we selected TMPRSS2 and MSPL facilitating PEDV replication in Vero cells without trypsin,and further confirmed the activation of TMPRSS2 and MSPL on S protein of PEDV promoting virions entry and cell fusion.We revealed the the molecular mechanisms affecting viral replication by the interaction of TTSPs with S protein of PEDV,providing an insight and and novel way for enhancing viral titers,expanding virus production and improving the adaptability of PEDV isolates in vitro.