Exploration And Functional Study Of EIAV Novel Protein

Equine infectious anemia virus(EIAV)is a member of lentivirus of retroviruses mainly infected horses,donkeys and other equine animals.EIAV and other lentiviral members such as human immunodeficiency virus(HIV-1)have similarities in virus morphology,genome composition,replication mechanisms,and interactions with the host.EIAV is considered to be an important model for studying the pathogenesis of lentivirus.Previous studies have identified EIAV as the simplest lentivirus with a genome comprising three structural protein genes(encoding Gag,Pol and Env,respectively)and three accessory protein genes(encoding Tat,S2 and Rev,respectively).In this study,we used PCR to identify a new mRNA transcript(Named s4)from EIAV infected tissues based on the transcriptional characteristics of the genome.The 5? splice donor site and 3? splice acceptor site of this transcript were different from other transcripts.It contained a complete open reading frame(ORF)(length 369 bp),which completely overlapped with the C terminal of the env and we named the protein encoded by this transcript S4.We identified s4 in many tissues such as bone marrow,liver,lymph nodes,brain of EIAV infected horses tissues,and in peripheral blood mononuclear macrophages of horses infected.S4 is about 18 kDa,mainly expressed in the cytoplasm.Moreover,we detected 18 kDa specific bands in EIAV infected horse macrophages using antiS4 protein polyclonal antibodies,which further suggesting that S4 proteins exist in EIAV infected cells.To study the role of S4 in viral replication,we constructed S4 expression-deletion EIAV infectious clones by reverse genetic techniques.Transfected it into HEK293 T cells found that the amount of virus particles in S4 expression-deleted EIAV in the supernatant was significantly lower than that in wild-type.However,it could significantly increase the release of the virus in HEK293 T cells transfected wild-type nd S4 expression-deleted type after overexpression of S4 protein.It indicating that S4 can regulate EIAV release.The innate immune restriction factor tetherin is an interferon-induced transmembrane protein,which exert its broad-spectrum antiviral activity by restricting the release of viral particles from the cell membrane.Previous studies have shown that human and equine tetherin can restrict EIAV release.To investigate how S4 regulate the release of EIAV,we transfected S4 expression-deletion EIAV infectious clones after interfering and knocking out the expression of tetherin in HEK293 T cells,we found that the release of viral particles in the supernatant was significantly increased.Moreover,we found that S4 could antagonize the restricting effect of equine tetherin on EIAV by co-transfected S4 and equine tetherin into HEK293 T cells.Thus,it indicating that S4 can antagonize tetherin to promote the release of viral particles.To explain the molecular mechanism of S4 antagonistic tetherin,we performed laser confocal and flow cytometry assays,the results revealed that S4 could significantly down-regulate tetherin expression on the membrane surface and sequester it to the endoplasmic reticulum and the trans-golgi apparatus by affecting its anterograde trafficking,but S4 did not affect the endocytosis of the tetherin.Taken together,this study identified a novel protein of EIAV for the first time,and confirmed that S4 promotes EIAV release by antagonizing antiviral activity of the tetherin.The results of this study will change the understanding of EIAV genome structure and demonstrate that EIAV contain at least 4 accessory proteins.