DOI: 10.1097/QAD.0b013e32833bfe3d

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PMID: 20622532

Issn Print: 0269-9370

Publication Date: 2010/07/31


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

Björn D Kuhl; Richard D Sloan; Daniel A Donahue; Tamara Bar-Magen; Chen Liang; Mark A Wainberg

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Author Information: McGill University AIDS Center, Lady Davis Institute, Jewish General Hospital, Canada

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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.
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