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. 2007 Nov 26:4:84.
doi: 10.1186/1742-4690-4-84.

Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages

Guillaume Jacquot  1 , Erwann Le Rouzic, Annie David, Julie Mazzolini, Jérôme Bouchet, Serge Bouaziz, Florence Niedergang, Gianfranco Pancino, Serge Benichou
Affiliations

Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages

Guillaume Jacquot et al. Retrovirology. .

Abstract

Background: HIV-1 Vpr is a dynamic protein that primarily localizes in the nucleus, but a significant fraction is concentrated at the nuclear envelope (NE), supporting an interaction between Vpr and components of the nuclear pore complex, including the nucleoporin hCG1. In the present study, we have explored the contribution of Vpr accumulation at the NE to the Vpr functions, including G2-arrest and pro-apoptotic activities, and virus replication in primary macrophages.

Results: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first alpha-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first alpha-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.

Conclusion: These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.

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Figures

Figure 1
Figure 1
Identification of Vpr mutants deficient for binding to the nucleoporin hCG1. A) Screening for Vpr mutants defective for the interaction with hCG1. The L40 yeast reporter strain expressing the wt or mutated (clones 11 and 35, and Vpr-R90K and -W54R single-point mutants) HIV-1 Vpr fused either to LexABD (upper panels) or to the Gal4 DNA binding domain (Gal4BD) (lower panels), in combination with each of the Gal4AD-hybrids indicated on the top was analyzed for histidine auxotrophy and β-Gal activity. Double transformants were patched on selective medium with histidine (+His) and then replica-plated on medium without histidine (-His) and on Whatman filters for β-Gal assay. Growth in the absence of histidine and expression of β-galactosidase indicated an interaction between hybrid proteins. B) Amino acid substitutions found in the hCG1-binding deficient Vpr mutants (clones 11 and 35). Mutants were derived by error prone PCR-mediated mutagenesis from the primary sequence of the VprLai strain that is shown at the top. C) Isolation of single-point Vpr mutants defective for the interaction with hCG1. Single-point mutants derived from Vpr clones 11 and 35 fused to LexABD were expressed in L40 strain in combination with each of the Gal4AD-hybrids indicated on the top. Double transformants were assessed as described in A).
Figure 2
Figure 2
Impact of the Vpr-L23F and -K27M substitutions on the three-dimensional structure of Vpr. A) 3D structure of HIV-1 Vpr [10], showing the three α-helices (residues 17–33, 38–50 and 54–77) represented in light blue, yellow and purple, respectively. The L23, K27, A30 and F34 residues are colored in red. The unstructured N- and C-terminal domains are represented in dark blue. B) CPK representation of Vpr. Residues are colored according to their hydrophobicity, except for L23 and K27 which are colored in yellow. The yellow box is enlarged in C), and this region shows a pocket that is organized around the L23 and K27 residues within the first α-helix and may represent a site for hCG1 binding. D) Helical-wheel diagram of the first α-helix of Vpr extending from a.a. D17 to F34. Residues L23, K27, A30 and F34 which have been mutated in the present study are indicated. Hydrophilic residues are in blue, whereas hydrophobic residues are in red.
Figure 3
Figure 3
Subcellular distribution of the Vpr mutants. A) Colocalization of Vpr and hCG1 at the NE. HeLa cells co-expressing Vpr-GFP (middle row) and Myc-hCG1 (left row) fusion proteins were permeabilized with digitonin, fixed, and subsequently stained with an anti-Myc monoclonal antibody. B and C) Localization of wt and mutated Vpr-GFP fusions. HeLa cells expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and directly examined. Cells were analyzed by epifluorescence microscopy, and images were acquired using a CCD camera. Scale bar, 10 μm.
Figure 4
Figure 4
G2-arrest and pro-apoptotic activities of the Vpr mutants. HPB-ALL T cells were transfected with the HA-tagged Vpr (wt or mutated) expression vectors in combination with the GFP expression vector. A) G2-arrest activity. The DNA content was analyzed 48 h after transfection by flow cytometry on GFP-positive cells after staining with propidium iodide. Results are expressed as the percentage of the G2M/G1 ratio relative to that of the wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. B) Pro-apoptotic activity. Cell surface PS exposure was analyzed 72 h after transfection by flow cytometry on GFP-positive cells after staining with phycoerythrin-labelled Annexin V. Results are expressed as the percentage of GFP-positive cells displaying surface PS exposure relative to that measured with wt HA-Vpr. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean. C) Expression of wt and mutated HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP (upper panels) and anti-HA antibodies (lower panels).
Figure 5
Figure 5
Subcellular localization of wild type Vpr and Vpr mutants in human monocyte-derived-macrophages. MDMs expressing either GFP, wt Vpr-GFP, or the indicated Vpr-GFP mutants were fixed and analyzed by wide-field microscopy. Z stacks of fluorescent images were acquired using a piezo with a 0.2 μm increment and one medial section is shown (left panels). Phase contrast images of the same cells were acquired to identify the nucleus (right panels). Scale bar, 5 μm.
Figure 6
Figure 6
Impact of the Vpr mutations on HIV-1 replication in monocyte-derived macrophages. A) Packaging assay of the wt and mutated HA-tagged HIV-1 vpr into virus like particles. 293T cells were transfected with an HIV-1-based packaging vector lacking the vpr gene in combination with vectors for expression of the wt or mutated HA-tagged Vpr protein. 48 h later, proteins from cell and virion lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-HA and anti-CAp24 antibodies. B and C) The L23F or K27M mutations were introduced into the vpr gene of the HIV-1YU-2 molecular clone. In B) Lysates from transfected 293T cells and virions isolated from cell supernatants were subjected to SDS-PAGE followed by Western blotting, using a rabbit polyclonal anti-Vpr and a mouse anti-CAp24 (provided from the NIH AIDS Research and Reference Reagent Program). In C) Replication of wild type and mutated HIV-1 in monocyte-derived macrophages. The wild type HIV-1YU-2 (WT, open diamonds) and the vpr-defective (ΔVpr, open squares), Vpr-L23F (black circles) and -K27M (black triangles) mutant viruses were produced by transfection of 293T cells with proviral DNAs. Monocyte-derived macrophages from four healthy donors were infected in triplicates with 0.5 ng of CAp24. Virus production was then monitored by measuring the p24 antigen by ELISA 10, 14 and 17 days after infection. Results are expressed as the level of p24 in the supernatants of infected cells. Values are the means of four experiments and error bars represent 1 standard deviation from the mean.
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