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