Interferon-stimulated Molecule Tetherin Inhibits EIAV Replication

Tetherin, also called CD317, BST-2or HM1.24, is a host natural immune factor, which is capableof blocking the release of enveloped viruses such as HIV-1. HIV-1is able to counteract tetherin’sfunction by its accessory protein Vpu. Human tetherin has broad antiviral activities to many otherretroviruses, which is considered as an interspecies barrier developed during long term interactionhistory between virus and host. Tetherin is inducible by type I Interferon (IFN-1). However, the mutualeffect between tetherin and EIAV which is the sole lentivirus that has been developed vaccine, has notyet been reported. In this paper, the inducible expression of tetherin and the inhibitory effect of humantetherin to EIAV are investigated.The eqIFNα1gene was amplified from the total RNA of equine spleen tissue, and cloned intoplasmid pVR-HA and used to transfect HEK293T cells. Western-blotting analysis showed that eqIFNα1was expressed in HEK293T cells, and was secreted to supernatant.In this study, the antiviral activity of eqIFNα1was detected by infecting equine dermal cells withVSV-GFP. The fluorescence microscope and flow cytometry observation indicated the dilution ofeqIFNα1has linear relationship with viral replication. The activity of eqIFNα1from supernatant iscalculated as1×105.5AU/mL.To identify whether eqIFNα1was able to inhibit EIAV release by inducing the expression oftetherin in HEK293T cells, we transfect EIAV GagPol construct in HEK293T cells to obtain equalexpression lever of EIAV Gag. Followed the transfection, eqIFNα1or control medium was added to thecells. The results showed eqIFNα1did not influence the level of GAGp55in HEK293T cells, but wascapable of decreasing the level of p26in the supernatant efficiently; a human tetherin specific real-timePCR was developed to detect huTetherin expression level. The huTetherin mRNA was increasedobviously in HEK293T cells treated with eqIFNα1. Co-transfection of EIAV GagPol and huTetherinshowed that the viral like particles (VLPs) in supernatant was decreased comparing with the control.These three assays demonstrated eqIFNα1is able to induce the expression of huTetherin largely, andhuTetherin can effectively inhibit the release of EIAV VLPs.Meanwhile, the relative real-time PCR-2-ΔΔCt, used to detect eqTetherin expression level, wasestablished and was used to detect the expression of eqTetherin in equine macrophage and equinedermal cells treated with eqIFNα1. The results indicated eqTetherin was inducible by eqIFNα1.The eqTetherin and huTetherin has the same ability in inhibiting the release of EIAV VLPs, whilehomologous rate between huTetherin and eqTetherin is only38%. So it is necessary to identify thefunction domain of eqTetherin. Therefore, mutants of delCT, delGPI, N51A, N78A were constructed.The results of co-transfection of these mutants and EIAV GagPol indicated that, compared with domainGPI, domain CT was more important for eqTetherin’s function; while two glycosylation sites, N51A andN78A, had no effect on eqTetherin’s function. Our results acquired gave detailed evidences that eqIFNα1has the cross-species antiviral activities,eqTetherin has similar structure basic to fulfill their function, and help to understand the antiviralmechanism of tetherin family.