Neither ritonavir nor darunavir affect cell surface expression of tetherin or Vpu-mediated tetherin down-modulation

Protease inhibitors act late in the HIV-1 life cycle, following viral assembly at the cellular membrane, to inhibit protease-mediated viral maturation. Virological outcome associated with the use of protease inhibitors is correlated with levels of pharmacokinetic exposure, which can be affected by drug metabolism and active removal of drugs from target cells [1]. Protease inhibitors serve as a substrate for several membrane-spanning drug transporters and efflux pumps [2,3]. Ritonavir (RTV) is a protease inhibitor that can increase both intracellular and extracellular concentrations of other protease inhibitors, partly by direct inhibition of drug transporters or efflux pumps, and is used as a boosting agent for other drugs in antiretroviral therapy [4,5]. Controversy exists as to whether RTV also interferes with calpain-mediated proteasomal degradation of drug transporters and other cell-membrane proteins. Whereas calpain was shown to be inhibited by RTV in cell culture models [6], these results were not confirmed in biochemical studies [7]. 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 [12]. 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[14]. 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. 1).Fig. 1: Tetherin cell surface expression levels are not significantly affected by protease inhibitors. Infected and uninfected cells were assessed for tetherin cell surface expression in the absence or presence of 5 μmol/l ritonavir (RTV), 10 μmol/l darunavir (DRV), or the combination of 5 μmol/l RTV/10 μmol/l DRV. Tetherin expression was induced by 0.1 μg/ml (a, c) or 1 μg/ml doxycyclin (dox) (b, d). Cell surface expression of tetherin was assessed by PerCP detection within Sup-T1 cell populations containing wt-infected (a, b) or Δvpu-infected (c, d) cells. Data are presented as geometric means; error bars represent the standard error of the mean.In summary neither RTV nor DRV appear to modulate tetherin levels at the surface of uninfected cells or of cells infected with wt or vpu-deficient virus. These results also imply that these protease inhibitors do not affect Vpu-mediated down-modulation of tetherin and that tetherin degradation is probably calpain-independent. Therefore, exposure to RTV is unlikely to lead to elevated levels of cell surface tetherin and it is unlikely that the antiviral activity of boosted protease inhibitor regimens can be attributed to any tetherin-related effects. Acknowledgements We are grateful to Professor Frank Kirchhoff for providing the viral clone pBR-NL43-IRES-eGFP and Dr Klaus Strebel for providing the hBST-2 antibody; both were provided through the NIH AIDS Research and Reference Reagent program. This research was supported by grants from the Canadian Institutes of Health Research.

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