Human immunodeficiency virus type 2 (HIV-2) Gag is trafficked in an AP-3 and AP-5 dependent manner

Although human immunodeficiency virus (HIV) types 1 and 2 are closely related lentiviruses with similar replication cycles, HIV-2 infection is associated with slower progression to AIDS, a higher proportion of long term non-progressors, and lower rates of transmission than HIV-1, likely as a consequence of a lower viral load during HIV-2 infection. A mechanistic explanation for the differential viral load remains unclear but knowledge of differences in particle production between HIV-1 and HIV-2 may help to shed light on this issue. In contrast to HIV-1, little is known about the assembly of HIV-2 particles, and the trafficking of HIV-2 Gag, the structural component of the virus, within cells. We have established that HIV-2 Gag accumulates in intracellular CD63 positive compartments, from which it may be delivered or recycled to the cell surface, or degraded. HIV-2 particle release was dependent on the adaptor protein complex AP-3 and the newly identified AP-5 complex, but much less so on AP-1. In contrast, HIV-1 particle release required AP-1 and AP-3, but not AP-5. AP-2, an essential component of clathrin-mediated endocytosis, which was previously shown to be inhibitory to HIV-1 particle release, had no effect on HIV-2. The differential requirement for adaptor protein complexes confirmed that HIV-1 and HIV-2 Gag have distinct cellular trafficking pathways, and that HIV-2 particles may be more susceptible to degradation prior to release

A new role for clathrin adaptor proteins 1 and 3 in lipoplex trafficking

Intracellular protein trafficking through secretory and endocytic pathways depends on the function of adaptor proteins that bind motifs on cargo proteins. The adaptor proteins then recruit coat proteins such as clathrin, enabling the formation of a transport vesicle. While studying the role of the clathrin adaptor proteins, AP-1, AP-2 and AP-3 in viral protein trafficking, we discovered that AP-1 and AP-3 potentially have a role in successful transfection of mammalian cells with DNA-liposome complexes (lipoplexes). We showed that AP-1, -2 and -3 are not required for lipoplexes to enter cells, but that lipoplexes and/or released DNA are unable to reach the nucleus in the absence of AP-1 or AP-3, leading to minimal exogenous gene expression. In contrast, gene expression from liposome-delivered mRNA, which does not require nuclear entry, was not impaired by the absence of AP-1 or AP-3. Despite the use of lipoplexes to mediate gene delivery being so widely used in cell biology and, more recently, gene therapy, the mechanism by which lipoplexes or DNA reach the nucleus is poorly characterised. This work sheds light on the components involved in this process, and demonstrates a novel role for AP-1 and AP-3 in trafficking lipoplexes

siRNA-mediated knockdown of AP-1 or AP-3 reduces GFP and METAFECTENE®-FluoR fluorescence in transfected cells.

<p>Quantitation of the experiments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091429#pone-0091429-g003" target="_blank">figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091429#pone-0091429-g004" target="_blank">4</a>. The percentage of GFP positive cells (<b>A</b>) and METAFECTENE®-FluoR positive cells (<b>B</b>) was counted at 6 hours (grey bars) and 24 hours (black bars) after GFP transfection. For each siRNA treatment at each time point, between five and nine fields of view (600–900 cells) from up to three independent experiments were analysed. Error bars represent the standard error of the mean. Asterisks indicate significant differences to no siRNA (−) samples (<b>A</b>, p = 5.7×10⁻⁵ and 3.1×10⁻⁴ for AP-1 and AP-3 siRNAs at 24 hours respectively; <b>B</b>, p = 0.013 and 0.026 for AP-1 and AP-3 siRNAs at 24 hours respectively).</p

AP-1 and AP-3 are not required for cellular uptake of lipoplexes.

<p>HeLa cells were transfected with no siRNA (−, <b>A</b>), siRNAs against AP-1γ, AP-2 µ or AP-3δ (<b>B–D</b> respectively) or a control siRNA (con,<b>E</b>). 24 hours later, the cells were transfected with GFP plasmid using a rhodamine B-labelled lipofection reagent, and fixed after 6 hours. Rhodamine fluorescence from METAFECTENE®-FluoR (red) is shown in the left panels, GFP fluorescence (green) is shown in the middle panels, and a merged image of the two channels is shown in the right panels. 25 µm scale bars are highlighted in the bottom right of the merged images.</p

siRNA-mediated knockdown of AP-1 or AP-3 reduces exogenous gene expression.

<p><b>A.</b> Relative luciferase activity of lysates from cells transfected with a <i>Renilla</i> luciferase plasmid 24 hours after siRNAs (grey bars) or co-transfected with plasmid and siRNAs (black bars). The data are from three or four independent experiments, error bars represent the standard error of the mean and asterisks indicate significant differences from no siRNA (−) samples (p = 9.5×10⁻⁵ and 5.1×10⁻⁴ for prior transfection of AP-1 and AP-3 siRNAs respectively). <b>B.</b> GFP fluorescence (green) of cells transfected with a GFP plasmid 24 hours after siRNAs. DAPI nuclear stain is shown in blue and 50 µm scale bars are shown in each image. The percentage of GFP positive cells was counted in ten fields of view (1000–2000 cells) for each siRNA treatment, from up to four independent experiments. The mean percentage of GFP positive cells relative to the no siRNA samples (−), +/− standard error, is shown. Asterisks indicate significant differences from no siRNA (−) samples (p = 2.7×10⁻¹¹ and 5.4×10⁻¹¹ for AP-1 and AP-3 siRNAs respectively).</p

siRNA-mediated knockdown of AP-5 affects HIV-2 but not HIV-1 Gag.

<p>HeLa cells were co-transfected with HIV-1 (A, B) or HIV-2 (C, D) proviral plasmids and no siRNA (none), siRNA against AP-5μ or a negative control siRNA (-ve). 42 hours post-transfection, cell lysates and viral supernatants were harvested. Cell-associated (A, C) and virion-associated (B, D) HIV-1 p55 (full length Gag), p41 (MA-CA) and p24 (CA), and HIV-2 p57 (full length Gag) and p27 (CA) were detected by western blotting (representative blots shown to the right) and quantified using densitometry. Error bars represent the standard error of the mean from three independent experiments and significant changes from no siRNA treatment are shown with an asterisk (p < 0.05).</p

siRNA-mediated knockdown of AP-1, -2 and -3 have different effects on HIV-1 and HIV-2 particle release.

<p>HeLa cells were co-transfected with HIV-1 (A, B) or HIV-2 (C, D) proviral plasmids and no siRNA (none), siRNAs against AP-1γ, AP-2μ or AP-3δ, or a negative control siRNA (-ve). 42 hours post-transfection, cell lysates and viral supernatants were harvested. Cell-associated (A, C) and virion-associated (B, D) HIV-1 p55 (full length Gag), p41 (MA-CA) and p24 (CA), and HIV-2 p57 (full length Gag) and p27 (CA) were detected by western blotting (representative blots shown to the right) and quantified using densitometry. Error bars represent the standard error of the mean from three independent experiments and significant changes from no siRNA treatment are shown with an asterisk (p < 0.05).</p

HIV-1 and -2 Gag differ in their subcellular distribution.

<p>Confocal immunofluorescence microscopy of HeLa cells transfected with envelope-deleted proviral plasmids. Gag immunofluorescence is shown in red, DAPI staining in blue. (A-C) HIV-1, (D, E) HIV-2. Scale bars are shown in the bottom right corner of each image. (F) Quantification of Gag distribution is shown graphically. Several hundred cells from three independent experiments were assessed by two different people. Cells were scored as having predominantly plasma membrane (PM), punctate intracellular (punctate), or diffuse cytoplasmic (diffuse) distribution of Gag. Error bars represent the standard deviation.</p

HIV-2 Gag localizes to CD63+/CD81+ compartments.

<p>Confocal immunofluorescence microscopy of HeLa cells (unless stated) transfected with proviral plasmids. Gag immunofluorescence is shown in red, DAPI staining in blue. (A) full length HIV-2 (expressing Env), (B) HIV-2 in 293T cells. (C) HIV-1, co-staining for Gag (red) and CD63 (green), (D) HIV-2, co-staining for Gag (red) and CD63 (green), (E) HIV-1, co-staining for Gag (red) and CD81 (green), (F) HIV-2, co-staining for Gag (red) and CD81 (green). Mean Gag and CD63 or CD81 co-localization (Pearson’s correlation coefficient, Rr) is shown to the right of images C and D or E and F respectively. Scale bars are shown in the bottom right corner of each image.</p

HIV-2 Gag co-localizes strongly with AP-3 and clathrin.

<p>Confocal immunofluorescence microscopy was carried out on HeLa cells transfected with HIV-1 (A-C, G-I) or HIV-2 (D-F, J-L) proviral plasmids. Gag is shown in red, AP-3 (B, C, E, F) or clathrin (H, I, K, L) are shown in green and DAPI is shown in blue. The overlay of the three channels is shown in the right hand image of each panel. Scale bars are shown in the bottom right hand corner of the overlay images. Mean Gag/AP-3 or Gag/clathrin co-localization (Pearson’s correlation coefficient, Rr) is shown to the right of each panel.</p