Abstract Background Tetherin (BST-2/CD317/HM1.24) is an interferon (IFN)-inducible factor of the innate immune system, recently shown to exert antiviral activity against HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface, thereby inhibiting viral release. In HIV-1 infection, the viral protein U (Vpu) counteracts this antiviral action by down-modulating tetherin from the cell surface. Viral dissemination between T-cells can occur via cell-free transmission or the more efficient direct cell-to-cell route through lipid raft-rich virological synapses, to which tetherin localizes. Results We established a flow cytometry-based co-culture assay to distinguish viral transfer from viral transmission and investigated the influence of tetherin on cell-to-cell spread of HIV-1. Sup-T1 cells inducible for tetherin expression were used to examine the impact of effector and target cell tetherin expression on virus transfer and transmission. Using this assay, we showed that tetherin inhibits direct cell-to-cell virus transfer and transmission. Viral Vpu promoted viral transmission from tetherin-expressing cells by down-modulating tetherin from the effector cell surface. Further, we showed that tetherin on the target cell promotes viral transfer and transmission. Viral infectivity in itself was not affected by tetherin. Conclusion In addition to inhibiting viral release, tetherin also inhibits direct cell-to-cell spread. Viral protein Vpu counteracts this restriction, outweighing its possible cost of fitness in cell-to-cell transmission. The differential role of tetherin in effector and target cells suggest a role for tetherin in cell-cell contacts and virological synapses.
Abstract Background Transcription of HIV-1 cDNA prior to, or in the absence of, integration leads to synthesis of all classes of viral RNA transcripts. Yet only a limited range of viral proteins, including Nef, are translated in this context. Nef expression from unintegrated HIV-1 DNA has been shown to reduce cell surface CD4 levels in T-cells. We wished to determine whether Nef expressed from unintegrated DNA was also able to downregulate the chemokine coreceptors CXCR4 and CCR5. Viral integration was blocked through use of an inactive integrase or by using the integrase inhibitor raltegravir. Infected cells bearing unintegrated DNA were assayed by flow cytometry in the GFP reporter cell line, Rev-CEM, for cell surface levels of CD4, CXCR4 and CCR5. Results In cells bearing only unintegrated HIV-1 DNA, we found that surface levels of CXCR4 were significantly reduced, while levels of CCR5 were also diminished, but not to the extent of CXCR4. We also confirmed the downregulation of CD4. Similar patterns of results were obtained with both integrase-deficient virus or with wild-type infections of cells treated with raltegravir. The Alu-HIV qPCR assay that we used for detection of proviral DNA did not detect any integrated viral DNA. Conclusions Our results demonstrate that Nef can be expressed from unintegrated DNA at functionally relevant levels and suggest a role for Nef in downregulation of CXCR4 and CCR5. These findings may help to explain how downregulation of CXCR4, CCR5 and CD4 might restrict superinfection and/or prevent signal transduction involving HIV-1 infected cells.
Abstract Background Integrase inhibitors are currently being incorporated into highly active antiretroviral therapy (HAART). Due to high HIV variability, integrase inhibitor efficacy must be evaluated against a range of integrase enzymes from different subtypes. Methods This study compares the enzymatic activities of HIV-1 integrase from subtypes B and C as well as susceptibility to various integrase inhibitors in vitro . The catalytic activities of both enzymes were analyzed in regard to each of 3' processing and strand transfer activities both in the presence and absence of the integrase inhibitors raltegravir (RAL), elvitegravir (EVG), and MK-2048. Results Our results show that integrase function is similar with enzymes of either subtype and that the various integrase strand transfer inhibitors (INSTIs) that were employed possessed similar inhibitory activity against both enzymes. Conclusion This suggests that the use of integrase inhibitors against HIV-1 subtype C will result in comparable outcomes to those obtained against subtype B infections.
MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to approximately 13% of wt levels and increased resistance to MK-2048 to approximately 8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer "off" rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg(2+) binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.
ABSTRACT Although transcription from unintegrated human immunodeficiency virus type 1 (HIV-1) DNA can occur inside infected cells, yielding all classes of viral mRNA transcripts, the translation of viral proteins is very limited. One of the proteins made is Nef, but it is unclear whether Nef produced in this way is able to play a role in immune evasion as occurs with integrated virus. We therefore asked whether transcription from preintegrated HIV-1 cDNAs could result in Nef-mediated modulation of cell surface major histocompatibility complex class I (MHC-I) expression. We infected a Rev-CEM green fluorescent protein (GFP) reporter cell line with virus and blocked integration though use of either an inactive integrase or the integrase inhibitor raltegravir. Infected cells were assayed by flow cytometry for cell surface expression of the HLA-A, HLA-B, and HLA-C allotypes (HLA-ABC), HLA-A31, and HLA-E. Viral RNA and DNA products were assayed via quantitative PCR (qPCR). The prevention of integration had no effect, relative to productively infected cells, on levels of expression of multiply spliced viral mRNA transcripts and Nef protein. Downregulation of HLA-ABC and HLA-A31 also occurred at levels similar to those seen in cells in which integration had occurred. Parallel experiments assaying cell surface HLA-ABC expression in infected activated primary CD4 + T cells produced a similar pattern of results. Hence, the capacity of HIV-1 to modulate MHC-I is not linked to its ability to integrate. Thus, Nef-mediated evasion of host immune responsiveness might be attributable, in part at least, to transcription from unintegrated viral DNA.
ABSTRACT HIV-1 can be transmitted as cell-free virus or via cell-to-cell contacts. Cell-to-cell transmission between CD4 + T cells is the more efficient mode of transmission and is predominant in lymphoid tissue, where the majority of virus resides. Yet the cellular mechanisms underlying productive cell-to-cell transmission in uninfected target cells are unclear. Although it has been demonstrated that target cells can take up virus via endocytosis, definitive links between this process and productive infection remain undefined, and this route of transmission has been proposed to be nonproductive. Here, we report that productive cell-to-cell transmission can occur via endocytosis in a dynamin-dependent manner and is sensitive to clathrin-associated antagonists. These data were obtained in a number of CD4 + T-cell lines and in primary CD4 + T cells, using both CXCR4- and CCR5-tropic virus. However, we also found that HIV-1 demonstrated flexibility in its use of such endocytic pathways as certain allogeneic transmissions were seen to occur in a dynamin-dependent manner but were insensitive to clathrin-associated antagonists. Also, depleting cells of the clathrin accessory protein AP180 led to a viral uptake defect associated with enhanced infection. Collectively, these data demonstrate that endosomal uptake of HIV-1 during cell-to-cell transmission leads to productive infection, but they are also indicative of a flexible model of viral entry during cell-to-cell transmission, in which the virus can alter its entry route according to the pressures that it encounters.
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.
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.
Background: Because of high intersubtype HIV-1 genetic variability, it has been shown that subtype-specific patterns of resistance to antiretroviral drugs exist. We wished to ascertain whether this might be true for integrase inhibitors. Methods: We compared the susceptibility of subtype B and C HIV-1 integrase enzymes, harboring the previously reported resistance mutations E92Q, N155H, and E92Q/N155H, to clinically relevant integrase inhibitors. This was performed biochemically using a microtiter plate system. Results: Subtype C integrase enzymes bearing the resistance mutations E92Q/N155H were approximately 10-fold more susceptible to each of two integrase inhibitors, raltegravir and elvitegravir, than were subtype B recombinant integrase containing the same mutations. Conclusion: Polymorphic differences within the subtype B and C integrase genes likely cause variations in the contribution of N155H alone or in combination with E92Q to drug resistance. It is possible that different viral subtypes may favor different mutational pathways, potentially leading to varying levels of drug resistance among different subtypes.
