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Overall, these results suggest that, under our experimental conditions, Vpu can downregulate intracellular and cell surface levels of BST2 in HIVinfected MDMs, with an intensity that differs between donors. Cellular and cell surface expressions of BST2 in infected macrophages. We therefore analyzed the impact of mutations of these motifs on the beneficial effect of Vpu on HIV-1 release in macrophages Fig.

This decrease was slightly less pronounced than that observed with Udel and A14W22 viruses. Moreover, this decrease was associated with an accumulation of cell-associated CAp24 Fig. Data were normalized to value for NL WT-infected cells. The P values are indicated above each comparison.

Interestingly, BST2 was proposed to contribute to the formation of VCCs 36 , although this hypothesis has been controversial In noninfected cells, BST2 labeling was detected as puncta dispersed throughout the cell, in a perinuclear compartment and at the cell surface; some BST2-positive puncta colocalized with CD81 Fig.

CD81 labeling is shown in gray in the monochrome panels to facilitate the visibility and in blue in the merge panels. Higher magnifications of virus-containing compartments VCCs are shown on the bottom left corner of each image.

CD81 labeling is shown in gray in the monochrome panels to facilitate visibility and in blue in the merge panels. Previous work showed a correlation between the presence of BST2 in VCCs and an increased size of these compartments sequestering viruses We thus determined the sum of the VCC volume per cell using three-dimensional 3D reconstruction.

Series of confocal images were then 3D reconstructed using Imaris software Fig. Interestingly, in a second set of experiments, we also observed a significant increase of the overall volume of these compartments in Vpu-mutated ELV The volumetric analysis of VCCs was performed using Imaris 7. An example of 3D reconstruction of MDMs is shown. The merge presented on the right is the overlap of the three two-dimensional 2D images corresponding to the 3D panels shown.

Arrows show structures considered VCCs. B and C Histograms showing the sum of the VCC volume per cell obtained by thresholding fluorescence intensity values on 62 cells under each condition from three independent donors. At 5 days postinfection, the cells were colabeled with anti-CAp24 and anti-BST2 antibodies and observed by confocal microscopy. As described above Fig.

A At 5days postinfection, cells were fixed, immunolabeled for BST2 green and CAp24 red , and analyzed by confocal microscopy. Higher magnifications inset of VCCs are shown on the right of each merge image. The histogram shows the sum of the VCC volume per cell obtained by thresholding fluorescence intensity values on at least 26 cells under each condition. However, in the absence of Vpu, these structures are enriched in BST2 and display an increased volume.

This volume relies on the lack of Vpu counteraction against BST2 activities. Finally, the increase of the VCC volume is not necessarily related to a strong enrichment of BST2 in these structures. This is consistent with the notion that BST2 does not contribute to the formation of VCCs but impacts their volume, depending on its capacity to tether viruses. In HIVinfected macrophages, newly synthesized virions are sequestered into subcellular virus-containing compartments VCCs 56 , — During cell-to-cell contacts, VCCs reach the cell surface and transfer viral particles to HIV-1 target cells, allowing HIV-1 to escape the host immune surveillance system The efficient spread of viral particles also depends on the ability of HIV-1 to circumvent intrinsic immunity, particularly the restriction factor BST2, which tethers fully formed viruses to the plasma membrane.

We notably highlight that the transmembrane domain of Vpu, as well as the E 59 XXXLV 64 motif and phosphoserine residues S 52 and S 56 in the cytoplasmic tail, is necessary for these functions. Using an HIV-1 mutant that is unable to express Vpu Udel virus , we confirmed previous reports 36 , — 38 , 51 , 60 revealing that Vpu is required for the efficient release of viral particles in primary human monocyte-derived macrophages MDMs Fig.

Interestingly, this upregulation of the BST2 protein level is observed at early time points of infection and is independent of HIV-1 Nef expression Fig. Our results did not sustain those presented by Chu and colleagues 36 and support a scheme in which the upregulation of BST2 expression is a consequence of the induction of type I IFN by infection rather than the expression of Nef protein Fig.

In these experiments, we noticed a significant variation of the HIV-1 Vpu response against BST2 restriction among the six donors analyzed. This singularity could explain the controversy observed in the literature regarding the impact of Vpu on the cell surface expression of BST2 in MDMs 36 , 38 , We also noted in our flow cytometry analysis that Vpu is unable to downregulate BST2 at the same level as that detected in noninfected macrophages in the majority of donors Fig.

These results agreed with those reported by Giese and Marsh 38 and are in line with previous reports indicating that Vpu-induced degradation of BST2 is not necessarily coupled with the ability of Vpu to promote HIV-1 release 37 , 44 , Previous studies have proposed that the volume of VCCs may correlate with the amount of accumulated viral particles 19 and that BST2 may indirectly favor the enlargement of these compartments by tethering viruses to the limiting membrane Our data obtained by 3D reconstruction of VCCs support these notions.

Indeed, we observed that Vpu expression induces a pronounced reduction of the total volume of these structures in HIVinfected macrophages Fig. These data corroborate the findings of Giese and Marsh but contradict the results reported by Chu et al. Infections of these cells with ELV or 2. Surprisingly, microscopy analyses revealed that the increased volume of the VCCs is not necessarily associated with a distinctive accumulation of BST2 in these compartments Fig. Consequently, these experiments point to a more complex contribution of Vpu functions to VCC formation and composition.

Therefore, we postulate that Vpu limits the size of VCCs by reducing the number of viruses tethered at the cell surface and, subsequently, sequestered into these structures once the plasma membrane invaginates. This phenomenon would be the result of the downregulation of BST2 molecules present at viral budding sites, through the interaction between the TM domains of Vpu and BST2, leading to the exclusion of BST2 from the budding sites.

Indeed, Vpu-induced degradation of BST2 is not strictly required for its induced direct exclusion of BST2 from viral budding sites 44 , — As BST2 is excluded from VCCs when cells are infected with these mutants, additional factors may contribute to virus entrapment. Interestingly, several additional factors, such as Siglec-1 and mannose receptor 1, have been described to tether virus and favor their sequestration in MDMs 17 , Such factors could participate in virus sequestration in VCCs, and Vpu could alter the cell surface expression of these factors and, in this way, modify virus sequestration in VCCs.

Thus, future work will be required to explore the role of additional cellular factors in virus sequestration in MDMs. Our study provides new insights into the mechanism regulating HIV-1 compartmentalization in infected macrophages. This process is based on the ability of Vpu to reduce the level of the restriction factor BST2 at viral budding sites. By this activity, Vpu reduces the accumulation of tethered virions in VCCs, preventing the expansion of these compartments.

Monocytes were obtained from HIV-seronegative human buffy coats. Mutagenesis and subclonings were verified by DNA sequencing. After protein separation, samples were transferred onto hydrophobic polyvinylidene difluoride PVDF membranes, followed by blocking in milk buffer Tris-buffered saline [TBS] [0. Blots were washed with TBS containing 0.

Forty-eight hours later, the cells were washed and infected. Cell culture supernatants were then collected and filtered on 0. At 5 days postinfection, MDMs were washed twice with ice-cold PBS, harvested by scraping, and pelleted by centrifugation. MDMs were washed three times, fixed, and analyzed as described above. To do so, 39 cells from 3 healthy donors under each infection condition were manually segmented by creating specific masks for the VCCs and the cytoplasm.

Measurement of the volume of the VCCs was allowed by 3D reconstruction of these compartments using Imaris 7. For each MDM, a z-stack of optical sections 0. Next, the 3D reconstruction of the VCCs was displayed using Imaris software, and the surface segmentation was manually adjusted to accurately select the VCCs. The values were obtained for at least 26 cells under each condition and from up to 3 independent donors depending on the experiment.

Alexa Fluor , Alexa Fluor , or Alexa Fluor conjugated secondary antibodies against mouse or rabbit purchased from Invitrogen and Cy5-conjugated anti-sheep antibody purchased from Jackson ImmunoResearch were used for immunofluorescence. Horseradish peroxidase HRP -conjugated secondary antibodies against mouse, rabbit, or goat immunoglobulin Dako were used for immunoblot experiments.

RNA concentrations were determined using a Nanodrop system. Analysis of each point was performed in technical duplicate. Strebel and A. Strebel and F. Maldarelli, and pNL from M. We thank K. J Virol. Published online May Prepublished online Mar Wesley I. Sundquist, Editor Wesley I. Sundquist, University of Utah;. Author information Article notes Copyright and License information Disclaimer.

Corresponding author. Address correspondence to Clarisse Berlioz-Torrent, rf. Contribution of the cytoplasmic determinants of Vpu to the expansion of virus-containing compartments in HIVinfected macrophages. J Virol e Received Jan 14; Accepted Mar 9. All Rights Reserved. This article has been cited by other articles in PMC. ABSTRACT HIV-1 infection of macrophages leads to the sequestration of newly formed viruses in intracellular plasma membrane-connected structures termed virus-containing compartments VCCs , where virions remain infectious and hidden from immune surveillance.

Open in a separate window. FIG 1. FIG 2. FIG 3. Vpu downregulates cellular and cell surface levels of BST2. FIG 4. The integrity of the Vpu transmembrane domain and of two motifs of the Vpu cytoplasmic domain is necessary for Vpu to promote HIV-1 release in macrophages. FIG 5. FIG 6. FIG 7.

FIG 8. FIG 9. Cell lines. Viral stocks. Western blot analysis. HIV-1 production. Flow cytometry analysis. Statistical analysis. Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus. J Med Virol 68 — Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J Exp Med — Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factortreated human monocytes: an ultrastructural study.

J Virol 62 — Antiviral Res 87 — Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses. Blood — Cell Host Microbe 16 — Tan J, Sattentau QJ. The HIVcontaining macrophage compartment: a perfect cellular niche? Trends Microbiol 21 — HIV-1 assembly in macrophages. Retrovirology 7 Electron microscopy analysis of viral morphogenesis. Methods Cell Biol 79 — Traffic 3 — Infectious HIV-1 assembles in late endosomes in primary macrophages.

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