Neither ritonavir nor darunavir affect cell surface expression of tetherin or Vpu-mediated tetherin down-modulation
Tetherin (BST-2/CD317/HM1.24) is an interferon-inducible integral membrane protein that contributes to innate cellular defense against infection by HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface and inhibiting viral release [8,9]. In HIV-1 infection, the viral protein Vpu counters this effect by promoting tetherin down-modulation from the cell surface as well as its subsequent endosomal/lysosomal or proteasomal degradation, leading to increased viral release [10,11]. Through its transmembrane domain, which also possesses an ion channel function, Vpu interacts with the transmembrane domain of tetherin [12,13]. The fact that RTV can block cell surface membrane exporters makes it important to understand whether this drug and other protease inhibitors might also impact on levels of tetherin expression and Vpu-mediated tetherin down-modulation in HIV-1-infected cells.
To investigate tetherin expression, we used Sup-T1 cells that contain the human tetherin gene . Tetherin expression was induced by adding either 0.1 or 1 μg/ml doxycycline (dox) (Sigma, St Louis, Missouri, USA). Doxycycline-induced cells were infected with either VSV-G-pseudotyped wt HIV-1 or with a Δvpu clonal derivative termed BR4-3-IRES-eGFP, which expresses enhanced green fluorescent protein (eGFP) from an internal ribosomal entry site downstream of nef. Induced Sup-T1 cells were infected to a percentage of 20–30%, as assessed by eGFP detection at 48 h postinfection (p.i.) by flow cytometry. Drugs were added at concentrations similar to those of plasma levels attained in patients receiving protease inhibitor drugs [darunavir (DRV), 10 μM; RTV, 5 μM; r/DRV, 5 μM RTV/10 μM DRV] [15,16]. Tetherin was stained using a rabbit antitetherin antibody with a secondary PerCP-labeled antirabbit antibody. Levels of cell surface tetherin were assessed by flow cytometry for PerCP at 48 h p.i. Uninfected and infected cells were distinguished by virus-derived eGFP expression. Data from at least three independent experiments were analyzed for statistical significance by one-way analysis of variance and Dunnett's post-test.
We found that cell surface tetherin levels, induced by 0.1 and 1 μg/ml dox, were significantly down-regulated in wt-infected cells compared to uninfected and Δvpu-infected cells under two different induction conditions (0.1 μg/ml dox, P = 0.03; 1 μg/ml dox, P = 0.02). This confirms that, in our system, tetherin down-modulation in HIV-1 infection is Vpu-dependent.
Moreover, we did not detect any statistically significant effect of the presence of either RTV, DRV, or both together on the cell surface presence of tetherin (0.1 and 1 μg/ml dox) in uninfected cells (0.1 μg/ml dox, P = 0.19; 1 μg/ml dox, P = 0.65) or cells infected with wt virus (0.1 μg/ml dox, P = 0.12; 1 μg/ml dox, P = 0.56) or Δvpu virus (0.1 μg/ml dox, P = 0.68; 1 μg/ml dox, P = 0.75) compared to controls not exposed to protease inhibitors (Fig.