The Prototype Foam Virus Uses Tas To Activate The NF-?B Signaling Pathway And Uses The NF-?B Factor To Enhance Virus Replication

Foamy virus is the only member of the Spumavirus.After the foam virus infects the host,the host will have a certain immune response,but there will be no obvious pathological damage,a persistent low-level infection state;however,PFV will have a cytopathic response when infected cells in vitro(Cytopathic effect(CPE),it is mainly due to the accum?lation of vacuoles in the cytoplasm and expansion of the endoplasmic retic?lum.The structure of the foam virus genetic,transcriptional reg?lation,life cycle and so on is also very different from other retroviruses.The NF-?B signaling pathway is one of the major immune reg?latory pathways.The NF-?B signaling pathway is mainly composed of p50(NF-?B1),p52(NF-KB2/p100),c-Rel(Rel),RelA,and Re1B.The NF-?B family of proteins can be functionalized by dimerization.The NF-?B signaling pathways include Classical pathway and Non-canonical pathway.The non-canonical NF-?B activation pathway is a replacement for the classical NF-?B activation pathway and a novel NF-?B activation pathway.After Activation of NF-?B factor that occur nuclear translocation and DNA binding induces target gene transcription.The NF-?B signaling pathway associated with apoptosis,viral replication,tumorigenesis,inflammation,and various autoimmune diseases.The virus also has a strategy to deal with the NF-?B signaling pathway,and it also uses certain factors of the host cell to accomplish or enhance its own replication.In order to verify that host cells are immunoregplated against viral invasion,we conducted the relationship between PFV and NF-?B signaling pathways.This experiment mainly analyzed the effect of the transactivator Tas of the foam virus on the host cell NF-?B and the effect of the host cell NF-?B signaling pathway on viral replication.The experimental res?lts obtained in this paper are as follows:(1)PFV transactivator Tas can activate NF-?B signaling pathway in host cellsFirst,the prokaryotic expression vectors of the three different hosts of the foam virus transactivator in the laboratory:pcDNA4-SF V-1-tas,pcDNA4-PFV-tas,pcDNA4-SFV-Ora-tas,and pNF?B-TA-luc,respectively.The luc reporter plasmid was simpltaneously transiently transfected into HeLa cells,and the activation activity of Tas for three different kinds of foam viruses against NF-?B was detected by a dual luciferase reporter system.Finally,the PFV transactivator Tas was found to be more active in NF-?B activation;the activity of NF-?B activation was compared between the two transactivators Bet and Tas in PFV.It was further verified that the PFV transactivator Tas can activate the NF-?B signaling pathway in the host cell.The res?lts showed that the transactivator Tas of the virus can activate the NF-?B signaling pathway in the host cell.(2)Detection of changes in NF-?B signaling pathway-related proteins.The expression of Tas protein was induced in Tas-induced expression cell line Hela PLVX-TRE3G-ZSGreen-Tas.After 48 hours,the transcription level and protein level of NF-?B signaling pathway-related genes in the stable expression of Tas protein were detected.Therefore,Tas expression After 48 hours,the expression of RelA and RelB protein in the nucleus was detected by Western Blot.At the same time,the total expression level of RelB was up-regulated,detecting RelA and RelB proteins in total protein,so Tas promoted the expression of RelB.On the other hand,we also visually detect RelA,P100 by immunofluorescence experiments,whether has a nuclear reaction under the action of Tas.Finally,immunofluorescence experiments and Western Blot changes demonstrated that Tas can activate NF-?B signaling pathway through classical,non-canonical pathways.And Tas can promote the up-regulation of RelB expression in non-canonical pathways.(3)Blocking the classical NF-?B activation pathway by mutating the I?Ba phosphorylation siteIn the previous experiments,we found that Tas can activate the NF-?B signaling pathway through classical and non-canonical pathways.The two pathways are composed of different factors.Therefore,in order to clarify which pathway is activated first,the serb and ser36 of I?B? are mutated to C.Amino acid blocks its phosphorylation,which in turn blocks the activation of the classical NF-?B pathway and detects changes in non-canonical pathway-associated proteins in this case.Although it has been reported in the literature that the classical NF-?B pathway may inhibit signaling on the non-canonical NF-?B pathway,it does not appear to have an effect on the non-canonical pathway after blocking the classical pathway.(4)Tas promotes viral replication along with the NF-?B signaling pathway RelBFurther,we found that the interaction of RelB and Tas can promote the transcription of viral LTR:the effect of RelA and RelB on LTR is detected by the dual luciferase reporter gene system.Analysis of the results indicated that RelB enhanced the transactivation activity of Tas compared to RelA and this enhancement was in a dose-dependent manner.If RelB is sequestered in the cytoplasm by p100,it is found that RelB attenuates the effect of Tas-induced LTR transcription.These data suggest that RelB interacts with Tas to promote viral replication.In summary,it was found that the prototype foam virus activates the NF-?B signaling pathway of cells using its transactivator Tas.This study clarified the relationship between the prototype foam virus and the NF-?B signaling pathway,and provided a theoretical basis for the further study of its latent infection mechanism.