Study On The Effect Of TEX2 On SVV Replication And Its Mechanism

Seneca Valley virus（SVV）,an emerging non-enveloped and single-stranded RNA virus belonging to the genus Senecavirus of the family Picornaviridae,caused a lot of attention due to its pathogenicity to pigs and oncolytic properties.The L^pro is the first protein of SVV encoded by the viral genome and plays a key role in the anti-host innate immune process.During infection with Erboviruses and Cardioviruses,L^pro acts as a potent novel virulence factor capable of repressing host antiviral responses.The SVV L^prolacks the catalytic residues necessary for proteolytic activity and does not contain a zincfinger motif,possibly indicating a function distinct from that L^pro of Aphthoviruses,Erboviruses and Cardioviruses.This study found that overexpression of SVV L^proinhibited SVV proliferation,which laid the foundation for further exploration of the function of SVV L^pro.Testis-expressed protein 2（TEX2）,also known as Transmembrane protein 96（TMEM96）,is mainly distributed in the endoplasmic reticulum membrane,plasma membrane and nuclear membrane,and contains two alpha helix transmembrane.This study found that TEX2 protein can inhibit the proliferation of SVV,provided a theoretical basis for the molecular mechanism of SVV replication,and provided a scientific basis for the development of anti-SVV drugs.Anthrax toxin receptor 1（ANTXR1）,also known as tumor endothelial marker 8（TEM 8）,was initially identified as the cell surface receptor of anthrax toxin,which is a transmembrane protein with extracellular domain.It is closely related to the process of bone formation and plays an important role in angiogenesis.It has been reported SVV-ANTXR1 binding has special significance because SVV was originally characterized as an oncolytic virus,which selectively kills tumor cells,does not replicate or kill normal human cells,and has good safety in clinical trials.ANTXR1 is used as a target for cancer treatment,as monoclonal antibodies,drug-conjugated antibodies and vaccines.ANTXR1 can also be used as a biomarker for different tumor types,including lung,breast and colorectal cancers.This study found that TEX2 can affect the replication of SVV.Further studies have found that TEX2 can interact with the SVV L^pro and SVV receptor ANTXR1,and further study its effect on viral proliferation and its mechanism.In view of this,the research carried out the following aspects:1.Study on the Influence of TEX2 on SVV ReplicationIn order to investigate the effect of transmembrane protein TEX2 on the replication of SVV,the human TEX2 gene was cloned into the eukaryotic expression vector p CAGGS-HA to obtain the recombinant plasmid p CAGGS-HA-h TEX2,transfected into293T cells,and infected with 0.1 MOI SVV 24 h later.The cells and supernatant were collected at 3、6、9 hpi,and the effects of TEX2 on SVV replication were evaluated by Western blot,real-time quantitative PCR（q RT-PCR）and viral plaque assay.The expression level of SVV VP1 protein in infected cells was detected by Western blot.The results showed that overexpression of TEX2 significantly inhibited the protein level of VP1.Real-time quantitative PCR results showed that the copy number of SVV VP1 gene in the TEX2 expression group was significantly lower than that of the control group（p<0.001）.Viral plaque assay results showed a significant decrease in viral titer of SVV in cells overexpressing TEX2 compared to the control group（p<0.001）.At the same time,the inhibitory effect of TEX2 on SVV proliferation was dose-dependent.To further determine the effect of TEX2 on SVV replication,the above results were verified on the basis of silenced TEX2.The effect of silencing TEX2 on SVV proliferation was evaluated by Western blot,q RT-PCR and viral plaque assay.The results showed that after down-regulation of endogenous TEX2 expression,viral protein VP1 levels,VP1gene copy number and virus titer were significantly increased,contrary to overexpression of TEX2.It was further confirmed that TEX2 can inhibit SVV replication.2.Study on the interaction between TEX2 and SVV L^proIn order to investigate whether the TEX2 protein inhibits the replication of SVV by interacting with the viral protein of SVV,the h TEX2 protein was co-transfected with the viral protein into 293T cells,and the Co-IP experiment was performed.The results showed that only could interact with TEX2,compared with the control group.Meanwhile,we found that TEX2 protein could degrade L^pro.To explore whether TEX2 has the ability to limit SVV replication by interacting with SVV L^pro,the eukaryotic expression plasmid p EBG-L^pro-GST was transfected into 293T cells.After 24 h,cells were infected with 0.1 MOI SVV,and cells and supernatant were collected at 3、6、9 hpi respectively.The effects of L^pro on the proliferation of SVV were evaluated by Western blot,q RT-PCR and viral plaque assay.The results showed that overexpression of L^pro inhibited SVV replication.This result indicated that TEX2 inhibits SVV replication not by interacting with L^pro.3.The effect of TEX2 on SVV entry and the impact of SVV infection on TEX2TEX2 is a transmembrane protein,laser confocal microscopy（LSCM）and cell membrane protein separation experiments show that TEX2 is almost entirely localized in the cell membrane.To explore the effects of TEX2 protein on SVV invasion of host cells,we performed viral attachment and internalization experiments.The results of real-time PCR and virus plaque assay showed that overexpression of TEX2 significantly down-regulated the copy number of SVV and decreased the titer of virus compared with the control group,indicating that TEX2 inhibits viral SVV through the entry stages of the virus replication.To evaluate the effect of SVV infection on endogenous TEX2 expression,293T cells were infected with 0.1 MOI SVV,and cell samples were collected at different time points.TEX2 protein expression and TEX2 m RNA expression were detected by Western blot and q RT-PCR.The results showed that the expression of TEX2 continued to increase with the prolongation of viral infection time.Therefore,we speculate that SVV may inhibit its proliferation due to the up-regulation of TEX2 protein expression.4.Study on the interaction between TEX2 and ANTXR1SVV is entried the cell by the receptor ANTXR1,in order to investigate whether TEX2 inhibits the proliferation of SVV by blocking the receptor ANTXR1.TEX2 and ANTXR1 were co-transfected into 293T cells.Co-IP experiments showed that TEX2protein can interact with ANTXR1 protein.Whether TEX2 inhibits the proliferation of SVV by blocking ANTXR1 remains to be further studied.This study demonstrates that the TEX2 protein interacts with the L^pro of SVV and can degrade L^pro,and reveals the effect of TEX2 on the proliferation of SVV and its molecular mechanism.These results allow us to understand the interaction between SVV and host proteins,and provide new ideas for subsequent SVV infection,immune research and antiviral drug design.