Host Protein Pals1 Inhibits Porcine Transmissible Gastroenteritis Virus Infection

Porcine transmissible gastroenteritis virus(TGEV)is a virus that causes intestinal diarrhea in pigs,belonging to the order of the Nest Virus,the Coronavirus family.TGEV infection is characterized by vomiting,diarrhea,dehydration,leading to 100% mortality in suckling piglets,causing huge economic losses to the pig industry around the world.Pals1 is a tight junction-related protein and a member of the CRB3-Pals1-Patj polar complex.These proteins are essential for the formation of cell junctions and the establishment of apical-basal polarity.On the one hand,tight junctions can make epithelial cells form a physical barrier,which plays an important role in the intestinal antiviral natural immune response.On the other hand,viruses have evolved the ability to degrade tight junction proteins to promote self-replication.Whether the tight junction protein Pals1 plays a role in TGEV replication has not been reported yet.It is necessary to systematically study the impact of Pals1 protein on TGEV replication.In order to study the role of Pals1 in virus infection,we first amplified and sequenced the gene of Pals1 in pig cells,then constructed p CMV-myc-Pals1 eukaryotic expression plasmid,and identified the ability of high expression in pig cells.We found that overexpression of Pals1 protein in cells can inhibit the replication of TGEV,while down-regulation of pals1 protein can promote the replication of TGEV.The results of the study indicate that the host cell Pals1 can inhibit the replication of TGEV.In order to study the effect of TGEV infection on Pals1,Western Blot was used to prove that the expression of pals1 was down-regulated after TGEV infection,which was time-and dose-dependent.Fluorescence quantitative technology proved that TGEV infection does not affect the m RNA level of Pals1,and only degrades Pals1 at the protein level.Through further drug inhibition experiments,we proved that TGEV infection down-regulates the expression of Pals1 through the ubiquitin-proteasome pathway.Subsequently,we used fluorescence microscope to observe that the subcellular localization of pals1 changed after TGEV infection and redistributed from the junction of cell membrane to the nucleus.Studies have shown that there is an antagonistic effect between viral infection and the Pals1 protein of host cells.In order to further study the interaction between viral infection and host Pals1 protein,we identified the viral proteins interacting with host cell pals1 by mass spectrometry.The results of the study preliminarily indicate that there may be interactions between the virus M protein and Pals1 in virus-infected cells.In order to further confirm this interaction,our study used the immunoprecipitation technique and found that Pals1 can immunoprecipitate TGEV M protein in virus-infected cells by using a commercial monoclonal antibody of Pals1.On the contrary,using monoclonal antibody against M protein,TGEV M protein can also immunize and precipitate pals1 in TGEV infected cells.The results of the study indicate that there is an endogenous interaction between the viral M protein and Pals1 in virus-infected cells.In summary,we found that on the one hand,the host Pals1 protein can inhibit the replication of TGEV,on the other hand,TGEV infection can degrade Pals1 and change the subcellular distribution of Pals1,we first discoverd Pals1 as a restriction factor blocking TGEV infection.In addition,in virus-infected cells,there is an endogenous interaction between the viral M protein and Pals1,which may play a role in the antiviral process.The experimental results of this study will provide new targets for the screening of antiviral drugs for TGEV.