siRNA screen for cellular factors required for HIV-1 and M-PMV assembly and release.

<p>(A) Schematic representation of the screen. siRNAs pools were transfected into HeLa or Cos-1 cells on day 1 in a 96-well format. A second transfection with the identical siRNA smartpool together with proviral vector was performed 24 hours after the first transfection. Following an additional 48 hour incubation, particle production in the supernatant was determined by reverse transcription assay. Intracellular GFP expression and cell viability as indicated by PI staining were also measured. (B) siRNA against TSG101 reduces HIV-1 virus output without significantly affecting cell viability and intracellular GFP levels. The scrambled siRNA or TSG siRNA was transfected into HeLa cells with NL4-3-EGFP HIV proviral vector as described above and the effects on HIV particle output, intracellular GFP expression and cell viability were quantified relative to control scrambled siRNA. Error bars indicate the standard deviation of three independent experiments. (C) siRNA against TSG101 reduces M-PMV virus output without significantly affecting cell viability and intracellular GFP levels. The scrambled siRNA or TSG siRNA was transfected into Cos-1 cells with pSARMX-EGFP and pTMO-Env expression vectors as described above and the effects on M-PMV particle output, intracellular GFP expression and cell viability were quantified relative to control scrambled siRNA. Error bars indicate the standard deviation of three independent experiments.</p

Effect of silencing of TSG101, ALIX1, AP1M1, BECN1 and SARA1 on HIV-1 and M-PMV particle output.

<p>(A) Quantitative western blots showing knockdown efficiency of the individual genes in Jurkat cells and Cos-1 cells. Jurkat cells and Cos-1 cells stably expressing control shRNA or specific shRNAs against individual genes were generated, and the cellular levels of the individual proteins examined by immunoblotting. The ratio of the individual protein levels as quantified on the LiCor Odyssey compared to control cell is shown below each blot. (B) The effect of depleting the individual genes on M-PMV particle output. Cos-1 cells stably expressing control shRNA or the indicated shRNAs were transfected with pSARMX M-PMV proviral vector. At 48 hours after transfection, the virions in the supernatant and cell lysates were harvested and subjected to immunoblotting with p27 antibody. The ratio of supernatant p27 and the ratio of the total amount of cellular p27 and Pr78^Gag as quantified on the LiCor Odyssey compared to control cells are shown below each blot. (C) The effect of depleting the individual genes on HIV-1 particle release in Jurkat cells. Control shRNA or specific shRNA expressing Jurkat cells were infected with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-NL4-3 virus (at 1 TCID₅₀) overnight at 37°C, washed and then seeded in 12-well plates. HIV-1 particle release was assessed using a p24 antigen ELISA 2 days post-infection. Results were expressed as percentage Gag release (supernatant p24/(supernatant + cellular p55/p24)) normalized to control shRNA-expressing cells. Error bars indicate the standard deviation of three independent experiments. (D) Quantitative analysis of the effect of depleting the individual genes on M-PMV particle release. M-PMV particle release in the control shRNA or specific shRNA expressing Cos-1 cells was assessed by immunoblotting with p27 antibody. The supernatant p27 and cellular p27 and Pr78^Gag were quantified on the LiCor Odyssey. Results were expressed as percentage Gag release (supernatant p27/(supernatant + cellular Pr78^Gag/p27)) normalized to control shRNA-expressing cells. Error bars indicate the standard deviation of three independent experiments.</p

Effect of silencing the selected genes on HIV1 particle output.

<p>(A) Quantitative western blot analysis of knockdown efficiency of the individual genes in HeLa cells. HeLa cells stably expressing control shRNA or specific shRNAs against each individual gene were generated and the cellular levels of the individual protein were examined. The ratio of the individual protein levels as quantified on the LiCor Odyssey compared to control cell is shown below each blot. (B) The effect of depleting the individual genes on HIV-1 particle output. HeLa cells stably expressing control shRNA or the indicated shRNAs were transfected with NL4-3 HIV proviral vector. At 48 hours after transfection, the virions in the supernatant and cell lysates were harvested and subjected to immunoblotting with p24 antibody. The ratio of supernatant p24 and the ratio of the total amount of cellular p24 and p55 as quantified on the LiCor Odyssey compared to control cells are shown below each blot.</p

Bioinformatics analyses of siRNA screen hits.

<p>(A) Venn diagram of siRNA hits identified in this work. We identified 41 host proteins in HIV screen and 52 host proteins in M-PMV screen. There were 24 host proteins that were common for both HIV-1 and M-PMV. (B) Protein interaction network of HIV1 screen hits (Stronger associations are represented by thicker lines). A map of the interactions of our HIV screen hits was built by using STRING 9.05 with a required medium confidence score (0.400). Analysis of the network revealed clusters of interrelated factors involved in three distinct pathways (highlighted in grey circle): cellular actin cytoskeletal regulation pathways, clathrin-mediated trafficking factors and regulators of phosphoinositide metabolism. (C) Protein interaction network of the common screen hits for both HIV-1 and M-PMV (Stronger associations are represented by thicker lines). The interactions map of 24 host proteins that were common for both HIV-1 and M-PMV was built by using STRING 9.05 with a required medium confidence score (0.400). Factors involved in clathrin-mediated trafficking, regulation of the actin cytoskeleton, and phosphoinositide metabolism (highlighted in grey circle) were identified to affect both HIV-1 and M-PMV assembly or release.</p

Effect of silencing the selected genes on M-PMV particle output.

<p>(A) Quantitative western blots showing knockdown efficiency of the individual genes in Cos-1 cells. Cos-1 cells stably expressing control shRNA or specific shRNAs against each individual gene were generated and the cellular levels of the individual protein were examined. The ratio of the individual protein levels as quantified on the LiCor Odyssey compared to control cell is shown below each blot. (B) The effect of depleting the individual genes on M-PMV particle output. Cos-1 cells stably expressing control shRNA or the indicated shRNAs were transfected with pSARMX M-PMV proviral vector. At 48 hours after transfection, the virions in the supernatant and cell lysates were harvested and subjected to immunoblotting with p27 antibody. The ratio of supernatant p27 and the ratio of the total amount of cellular p27 and Pr78^Gag as quantified on the LiCor Odyssey compared to control cells are shown below each blot.</p

Depletion of CAML in HeLa cells fails to relieve restriction to particle release.

<p>A stably-transduced cell population demonstrating marked CAML knockdown (lanes 3, 4, 7, 8) was transfected with NL4-3 or NLUdel proviral DNA and compared with control shRNA transduced cells (lanes 1, 2, 5, 6). CAML protein is shown in red. In the presence of Vpu, particle release was equivalent in CAML knockdown or control cells (lanes 1–4). In the absence of Vpu, the restriction to particle release was present and was not affected by CAML depletion (lane 8 vs. lane 6).</p

CAML does not regulate cell surface levels of tetherin.

<p>A rabbit polyclonal anti-tetherin antibody was used to quantify tetherin on the cell surface of HeLa cells by flow cytometry. A) Unstained cell control (white) and tetherin surface staining (filled histogram) using primary rabbit anti-tetherin followed by anti-rabbit APC staining. B) HeLa cells transduced with a control shRNA lentiviral vector (filled histogram) compared with unstained control cells (white). Dashed line indicates histogram of tetherin shRNA-transduced cells, demonstrating that cell surface levels were diminished by tetherin shRNA. C) Cell surface levels of tetherin in control shRNA transduced cells (filled histogram) did not differ from cells in which CAML was significantly depleted using CAML-specific shRNA (dashed lines). D) Vpu-EGFP transfected HeLa cells demonstrate cell surface downregulation of tetherin, as indicated by left shift of EGFP-positive population. E) CAML knockdown HeLa cells demonstrate cell surface downregulation of tetherin by Vpu-EGFP, similar to that shown in D.</p

Direct comparison of CAML and tetherin effect on particle release in 293T cells and HT1080 cells.

<p>A) 293T cells were transfected with increasing amounts of CAML or tetherin expression vectors and NL4-3 (left) or NLUdel provirus (right). Cells and particles in supernatants were harvested at 48 hours post-transfection and analyzed by Western blotting using an anti-p24 monoclonal antibody. Total amount of transfected CAML or tetherin plasmid DNA per 35 mm² well is indicated above the blots. B) HT1080 cells were employed in experiments identical to those described for 293T cells. No consistent effect of CAML on virus release was noted in either cell type, while tetherin potently inhibited virus release. Molecular mass markers are indicated on the left of each panel.</p

CAML depletion does not inhibit tetherin-mediated restriction of particle release.

<p>293T cells were transduced with an shRNA-encoding lentivirus expressing control shRNA or CAML shRNA and selected with puromycin. Selected cell populations were then transfected with tetherin expression vector and NLUdel proviral DNA. Cells (C) and supernatants (S) harvested at 48 hours for Western blot analysis. Particles present in supernatants were pelleted through 20% sucrose prior to loading. Analysis was performed using infrared detection, allowing simultaneous assessment of CAML in cell lysates by rabbit polyclonal antisera (red) and HIV Gag proteins by anti-p24 monoclonal antibody (green). Co-transfection of cells with a tetherin expression construct is indicated by plus signs. M  =  molecular mass markers.</p

Tetherin is interferon-inducible; CAML is not interferon-inducible.

<p>A) Western blot for endogenous tetherin was performed using rabbit polyclonal antisera before or 48 hours after interferon-induction. Note that induction was demonstrated in each case, and baseline levels of tetherin were highest in HeLa cells. Lanes were normalized by total protein quantitation. D) Western blotting for endogenous CAML was performed in the same panel of cells. Note that no induction was demonstrated, and all cell types expressed detectable CAML. Cell lines are indicated above the blots, and IFN treatment is indicated with a plus sign. M  =  molecular mass markers.</p