Abstract
Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.
Highlights
The human immunodeficiency virus 1 (HIV-1) has a complex retroviral genome, which, in addition to encoding the classical structural and enzymatic proteins Gag, Gag-Pol, Pol and Env, and the regulatory proteins Tat and Rev, encodes the four accessory proteins Vpr, Vif, Vpu and Nef that play multiple roles in HIV-1 pathogenesis
Cells were cultured in media containing BIT225 at concentrations of 0.04, 0.2, 1, 5, 10, 25 or 50 mM for 72 h, after which we assessed cell viability using flow cytometry detection of side scatter (SSC) and forward scatter (FSC), comparing BIT225 treated populations to dimethyl sulfoxide (DMSO) treated controls (Figure S1)
We assessed the inhibitory effect of BIT225 on virus release in these cell lines infected with equal amounts of wt or Dvpu virus and cultured in media containing BIT225 at concentrations of 0.04, 0.2, 1, 5, 10, 25 or 50 mM at 72 h post infection (p.i.) (Fig. 1, Table 1)
Summary
The human immunodeficiency virus 1 (HIV-1) has a complex retroviral genome, which, in addition to encoding the classical structural and enzymatic proteins Gag, Gag-Pol, Pol and Env, and the regulatory proteins Tat and Rev, encodes the four accessory proteins Vpr, Vif, Vpu and Nef that play multiple roles in HIV-1 pathogenesis (reviewed in [1,2]). The viral protein Vpu is a 16 kDa type I transmembrane protein, consisting of a N-terminal transmembrane domain (AA 1– 27) and a cytoplasmic domain (AA 28–81) of two consecutive amphiphatic a-helices (AA 33–49 and AA 57–70) [2,10,11]. The most studied function of Vpu is the downmodulation of CD4, which permits Env trafficking to the viral assembly site and subsequent incorporation into the viral membrane. This CD4 downmodulation occurs in the endoplasmic reticulum (ER) and is mediated by the C-terminal domain of Vpu acting as a transient adaptor protein to link CD4 to b-transducin repeats-containing protein (b-TrCP), resulting in proteasomal degradation of CD4 but not of Vpu (reviewed in [14])
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