Effect of tetherin from different hosts on virus release.

<p>A. Virus particle release was tested in 293T cells transfected with HIV-1ΔVpuΔNef NL4.3 proviral DNA (500 ng) in the presence of human, <i>C. denti</i> and <i>C. neglectus</i> tetherin. Virus production expressed as a percentage of maximal particle release in the absence of tetherin is shown for increasing amounts of plasmid DNA encoding for the three tetherin constructs (100, 330 and 1000 ng). Differences in the amount of plasmid DNA in each transfection were compensated by the addition of control vector (pcDNA3). After 42 h, the amount of virus released into cell culture supernatant was measured by HIV-1 p24 ELISA. This figure is representative of three independent experiments. B. Expression of Vpu constructs. Western blot analysis was performed to determine the expression levels of the three plasmids encoding HA-tagged tetherins. 293T cells were cotransfected with 1000 ng of each plasmid. Tetherin migrated as several species in SDS-PAGE analyses, presumably as a result of heterogeneus glycosylation. Forty-two hours after transfection cell lysates were collected and analyzed by Western blot. The expression level of HIV-1 Gag protein was monitored with an anti-p24 antibody. Actin was used as a loading control.</p

Virion release from cells tranfected with vectors expressing tetherin mutants.

<p>HIV-1 Vpu and SIVden Vpu were tested for their ability to rescue p24 release for HIV-1ΔVpuΔNef in 293T cells expressing human tetherin delta 25–26 (A) or <i>C. denti</i> tetherin insertion 28–29 (B). Twenty-four h after transfection the p24 content of the supernatant from each sample was quantified using a p24 ELISA. and expressed as the percent of p24 of the control without tetherin. Results are the mean ± SD for 5 independent experiments. The indicated p values correspond to the comparison with the «no vpu» sample.</p

Alignment of tetherin amino acid sequences.

<p>A. Alignment of the cytoplasmic tail and transmembrane domains of tetherins from 16 primates. The accession numbers of the sequences used in this figure are: Human NP 004326; <i>Gorilla gorilla</i> ADI58594; <i>P. troglodytes</i> ADI58593; <i>P. paniscus</i> ADI58595; <i>C. nictitans</i> ACX46125; <i>C. mona</i> ACX46126; <i>C. cephus</i> ACX46508; <i>P. nemaeus</i> ADI58604; <i>C. aethiops</i> ADI58600; <i>E. patas</i> ADI58599; <i>P. pygmaeus</i> ADI58596; <i>M. mulatta</i> ADI58602; <i>N. leucogenys</i> ADI58597; <i>H. agilis</i> ADI58598; <i>C. guereza kikuyuensis</i> ADI58603; <i>M. talapoin</i> ADI58601. B. Differences in the TM domain of human, <i>C. denti</i> and <i>C. neglectus</i> tetherins are highlighted in the blue box. Red numbers and letters indicate the amino acids we mutated in this study. In both panels, amino acid identity is indicated by dots and sequence gaps are indicated by dashes.</p

SIV Vpu does not reduce total tetherin levels but leads to depletion of tetherin from the cell surface.

<p>(A) 293T cells were cotransfected with HIV-1ΔVpuΔNef proviral construct (500 ng) and a plasmid encoding HA-tagged tetherins (human (100 ng), <i>C. denti</i> (1000 ng) and <i>C. neglectus</i> (1000 ng) and with or without HIV-1 Vpu plasmid (250 ng) or SIVden Vpu plasmid (250 ng). The effect of the two Vpu constructs on tetherin protein levels was monitored by Western blotting, using an anti-HA antibody. Tetherin migrated as several species in SDS-PAGE analyses, presumably as a result of heterogeneus glycosylation. The depicted gel is representative of three independent experiments. (B) Subcellular localization of tetherin. 293T cells were cotransfected with 1000 ng of DNA encoding the indicated tetherin proteins (HA-tagged), with a proviral construct (1000 ng) and with a HIV-1 Vpu or SIV Vpu plasmid (500 ng). 24 h after transfection the cells were fixed, permeabilized, and stained with rat anti-HA (green) monoclonal antibody (clone 3F10, Roche Applied Science). Nuclei are stained with DAPI (blue). Cells were examined by confocal microscopy. Images are representative of three independent experiments. Scale bars represent 10 µm.</p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-5

Ted monoclonal antibodies or isotype-matched control antibodies conjugated with the same flurochrome. (A) CD4 expression on U373-R5 cells (thin solid line) and MT4-R5 cells (heavy solid line) detected using FITC-labelled mouse anti-human CD4 monoclonal antibody (clone RPA-T4). Binding of an isotype-matched control antibody conjugated with FITC is shown in the corresponding dashed lines. (B) CCR5 expression on U373-R5 cells (thin solid line) and MT4-R5 cells (heavy solid line) detected using phycoerythrin-conjugated mouse anti-human CCR5 monoclonal antibody (clone 2D7). Binding of an isotype-matched control antibody conjugated with phycoerythrin to these cells is shown in the corresponding dashed lines.<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-0

F encompassing C1 to V2 (corresponding to nucleotides 6440 – 6809 of HXB2) of the 70 clonal viruses evaluated in the study (open circles, strict R5 tropism, solid black circles, strict X4 tropism; solid gray circles, dual tropism), the consensus sequence of the same region for viral RNA amplified by RT-PCR from an aliquot of the plasma sample used to generate the clonal viruses (stars), and the sequence of the laboratory strain pNL4-3. Bootstrap values are also indicated. For patient 2, the consensus sequence of plasma viruses grouped with clonal viruses with X4 tropism at M26, and with clonal viruses with R5 tropism at M34.<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-3

Ty of these viruses to infect MT4-R5 cells was measured by evaluating luciferase expression in the target cells 44 hours after infection. For patient 2, clonal viruses were obtained from plasma samples obtained 8 months apart (26 and 34 months after initial diagnosis). The tropism of the recombinant viruses, as defined by their ability to infect U373-R5 and U373-X4 cells is shown: strict R5, open symbols; dual, solid gray symbols; strict X4, solid black symbols. Results for viruses expressing sequences amplified by RT-PCR from patient plasma is also shown (stars). Each symbol is the mean of at least 3 independent experiments; the mean coefficient of variation for these results is 37% (range 1 – 78%). For each patient, significant differences were found comparing the viruses with the highest and lowest infectivity (p < 0.05 – 0.005 by t-test). (B) For each recombinant virus, the infectivity [(RLU/sec)/(ng p24/ml)] observed using U373 target cells and MT4-R5 target cells is expressed as a ratio.<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-2

Es), patient 5 (squares) or from the laboratory-adapted strain NL-AD8 (diamond), two types of recombinant reporter viruses were generated, one in which gp120 + only the extracellular domain of gp41 was derived from the clonal virus (pNL4-3-ΔectoENV-lucR, abscissa), and one in which gp120 and all of gp41 was derived from the clonal virus (pNL4-3-ΔENV-lucR vector, ordinate). The ability of these viruses to infect U373-R5 cells was compared by evaluating luciferase expression in the target cells 44 hours after infection. The infectivity of each pair of viruses was evaluated in three independent experiments, and each symbol represents the mean of these determinations. The dotted line is the line of identity. The correlation coefficient (Spearman) for the data shown is 0.86 (p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-7

Ed by the 17 clonal viruses from patient 2 with strict R5 tropism are shown. The consensus amino sequence is shown on the top line, and only amio acids differing from the consensus sequence are shown for each clone. For each variable region, sequences that are identical or that differ by a single amino acid substitution not identified in another sequence are highlighted by the same color, and these haplotypes are also identifed by numbers adjacent to the brackets. The red arrow indicates the envelope expressing the V2 region haplotype 4 associated with the V3 region haplotype 1 (see text). (B) The infectivity of recombinant viruses expressing these Env proteins using U373-R5 cells is shown. For each of the variable regions, the color of the symbols corresponds to that of the V region haplotype expressed by that virus. The red arrow indicates the infectivity of the clone expressing the V2 region haplotype 4 associated with the V3 region haplotype 1.<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p

Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses-4

F these viruses to infect MT4-R5 cells and U373-R5 cells was measured by evaluating luciferase expression in the target cells 44 hours after infection. For each of the 53 viruses with strict R5 tropism, the infectivity ratio (infectivity for U373-R5 cells/infectivity for MT4-R5 cells) is expressed as a function of the infectivity for MT4-R5 cells [(RLU/sec)/(ng p24/ml)]. A significant inverse correlation between these parameters was observed (Spearman r = -0.64, p < 0.0001).<p><b>Copyright information:</b></p><p>Taken from "Functional diversity of HIV-1 envelope proteins expressed by contemporaneous plasma viruses"</p><p>http://www.retrovirology.com/content/5/1/23</p><p>Retrovirology 2008;5():23-23.</p><p>Published online 29 Feb 2008</p><p>PMCID:PMC2270869.</p><p></p