Description of patients and <i>vpr</i> alleles analyzed in the study.

<p>A) Immunological and viral profiles of the patients analyzed. These patients were selected from the HIV-1-infected patient cohort of the St-Antoine Hospital (Paris), and the samples were collected with written informed consent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone.0007514-Lefrere1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone.0007514-Lefrere2" target="_blank">[46]</a>. The graphs show the time-course evolution of the blood CD4+ T cell counts (green curves) and plasmatic virus load (viral RNA, red curves); the PBMC samples analyzed in the present study are indicated by the blue arrows. B) Alignment of the amino acid sequences derived from DNA sequencing of the <i>vpr</i> alleles cloned from PBMC DNA samples. Excepted for LTNP-05 and LTNP-07 sequences that were obtained by direct sequencing of the bulk PCR fragments amplified from these samples, at least 40 independent clones were sequenced from each other samples. The sequences of the primary Vpr proteins are aligned with respect of the prototypic HIV-1<i>Lai</i> sequence (upper sequence). The <i>vpr</i> alleles from the LNTP (patient 5071) that were selected for subsequent functional analysis are in the box; the R77Q substitution identified in some <i>vpr</i> alleles is indicated in blue, while the Q65R substitution identified in the Vpr LTNP-04-2 is indicated in red.</p

G2-arrest and pro-apoptotic activities of Vpr proteins from the LTNP and PR patients.

<p>HPB-ALL T lymphoid cells were co-transfected with vectors for expression of HA tagged Vpr<i>Lai</i> or the indicated primary Vpr proteins from the LTNP and PR patients, in combination with the GFP expression vector. A) Cellular expression of HA-tagged Vpr proteins. Lysates from HPB-ALL transfected cells were analyzed by western-blotting using anti-GFP and anti-HA antibodies. B) G2-arrest activity of primary Vpr proteins. 48 h after transfection, cells were fixed and the DNA content of GFP-positive cells was analyzed by flow cytometry after DNA staining with propidium iodide. Results are expressed as the G2M/G1 ratios relative to that of the VprLai. C) Pro-apoptotic activity of Vpr proteins. Cells co-expressing the GFP and HA-tagged Vpr proteins were assayed by flow cytometry 72 h following transfection for cell surface phosphatidylserine exposure using AnnexinV coupled to phycoerythrin. Values are means of three independent experiments; error bars represent one standard deviation from the mean.</p

Interaction of the Vpr proteins from the LTNP and PR patients with hCG1, UNG2 and DCAF1 in the yeast two-hybrid system.

<p>The L40 reporter yeast strain expressing Vpr<i>Lai</i> or the indicated primary Vpr proteins from the LTNP and PR patients fused to LexA (LexA hybrid), in combination with either hCG1, UNG2 or DCAF1 fused to the Gal4 activation domain (Gal4AD hybrid), was analyzed for histidine auxotrophy. Growth in the absence of histidine indicates interaction between hybrid proteins.</p

Impact of the residue 77 on pro-apoptotic and G2-arrest activities of Vpr LTNP variants.

<p>The Arg77 residue from VprLai and Vpr LTNP-96 was replaced by a Gln, whereas the Gln residue from LTNP-86 and LTNP-04-1 was replaced by an Arg. The wild-type (grey bars) and mutated (hatched bars) Vpr proteins were then analyzed for apoptosis (A) and G2-arrest (B) activities as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g004" target="_blank">Figure 4</a>.</p

Relative frequency of primary Vpr alleles^a.

a<p><i>vpr</i> genes were amplified by PCR from each PBMC sample (indicated by blue arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g001" target="_blank">Fig. 1A</a>), cloned into a shuttle plasmid, and DNA sequences from at least 40 independent clones were determined from each sample.</p>b<p>samples 1986, 1996 and 2004 are from the LTNP patient (5071), and sample PR is from the progressor patient (1200).</p>c<p>Vpr alleles correspond to the primary amino acid sequences reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007514#pone-0007514-g001" target="_blank">Fig. 1B</a>.</p

Cellular localization of Vpr proteins from the LTNP and PR patients.

<p>(A) Co-localization of VprLai and hCG1 at the NE. HeLa cells were transiently transfected with vectors for expression of VprLai fused to GPF together with hCG1 fused to the Myc tag. 16 h after transfection, cells were fixed and subcellular distribution of the fusion proteins was analyzed by epifluorescence microscopy. (B) Subcellular distribution of the indicated primary Vpr proteins from the LTNP and PR patients fused to GFP. Cells expressing GFP were used as a control.</p