A pilot study on interaction between donkey tetherin and EIAV stains with different virulent and replication characteristics
Introduction
Recent studies have extensively characterized a number of host restriction factors that restrict viruses at different stage during infection [1], [2], [3], [4], [5]. Tetherin was first identified by Neil et al. (2008) [6], who firstly characterized its ability to restrict the release of HIV-1 from the cell surface. Subsequently, more evidence demonstrates that tetherin exerts a broad antiviral effect on various viruses [7], [8], [9]. Indeed, many viruses encode viral proteins to antagonize the restriction mediated by tetherin through different strategies [10], [11], [12], [13], [14], [15].
Equine infectious anemia virus (EIAV) has the simplest genome structure among all lentiviruses [16], which makes it an ideal model system in which to study the role of specific viral genes in lentiviral replication and persistent infection [17]. Our laboratory has previously developed stably attenuated virulent EIAV strains using a specific passage attenuation system [18], [19]. The major strains generated by this process were EIAVDV, EIAVDLV, and EIAVFDDV, each of which exhibit significant differences in virulence and replication both in vivo and in vitro.
Although equine and donkey tetherin were known to inhibit EIAV release from infected cells, it is unclear whether the arms race between tetherin and EIAV influences either the dynamic evolution of these EIAV strains or the attenuation of their virulence during the adaptive evolution process. To address this question, we evaluated the interplay between do-tetherin and these three different EIAV stains (EIAVDV, EIAVDLV, and EIAVFDDV). Interestingly, we found that do-tetherin has a different antiviral effect on virulent vs. attenuated EIAV strains. Additionally, Env proteins of all three EIAV strains could antagonize the inhibition mediated by do-tetherin in different extent. These results advance our understanding on the interplay between host restriction factors and EIAV evolution.
Section snippets
Cell culture
Human embryonic kidney (HEK) 293T cells were maintained at 37 °C in a 5% CO2 incubator in Dulbecco's Modified Eagle's Medium (HyClone, Utah, USA) supplemented with 10% fetal bovine serum (SAFC, Buchs, Switzerland) and 1% penicillin/streptomycin (100 units/ml, HyClone, Utah, USA).
Construction of expression plasmids
EIAVDV, EIAVDLV, and EIAVFDDV were grown in culture, and total RNA was extracted separately from each culture medium using a QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Germany). cDNA was then synthesized using a Reverse
Do-tetherin inhibited all three EIAV strains with varying degrees of strength
To determine whether do-tetherin could restrict the budding of virulent strains of EIAV, do-tetherin was co-transfected with the GagPol-DV, GagPol-DLV, or GagPol-FDDV plasmid into 293T cells. After 48 h, the cells and the supernatant were harvested and subsequently analyzed by western blot. The results demonstrated that the release of the EIAV VLPs was inhibited by do-tetherin in a dose-dependent manner (Fig. 1A and B). In addition, do-tetherin had varying effects on the budding of virulent vs.
Discussion
Attenuated EIAV strains have been developed in our specific EIAV attenuation system through a lengthy in vitro culturing and passaging process [19], with dynamic interactions between the virus and host occurring throughout this process. Because there is no adaptive immune response in this in vitro culturing process, we hypothesized that the innate immune response of the host cells, particularly the stimulated restriction factors, is involved in the attenuation of viral virulence. As an
Conflict of interest
The authors have no financial conflicts of interest to report.
Acknowledgments
This study was supported by a grant from the National Natural Science Foundation of China (31222054; X-J W).
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These authors contributed equally to this article.