Mechanisms of anti-HIV activity of CAP treatment.

<p>A1. MDM treated with CAP or helium (control) were analyzed by flow cytometry for expression of CCR5 and CD4. A2. Results are presented as mean ± SEM for analysis performed for MDM from two donors. P values (relative to control) were calculated using Student’s unpaired two-tail <i>t</i>-test. B. Fusion between HIV-1 and MDM was analyzed by fluorescence resonance energy transfer-based fusion assay. Cleavage of CCF2 represents virus-cell fusion. C. MDM treated with CAP or helium (control) were infected in triplicate wells with HIV-1 ADA (R5 virus) or VSV-G-psudotyped HIV-1 NL4-3 (X4 virus), and viral production was measured on day 7 by RT activity. Results are presented as mean ± SD. *p = 0.0037, **p = 0.027 relative to control, calculated using Student’s unpaired two-tail <i>t</i>-test. D. MDM treated with CAP or helium (control) were infected with HIV-1 ADA and incubated for 21 days. Virus was collected, adjusted to the same RT activity by dilution, and used to infect indicator TZM-bl cells. Results are presented as mean ± SD for 5 independent replicates. ****p<0.0001 by Student’s unpaired two-tail <i>t</i>-test.</p

Analysis of CAP cytotoxicity.

<p>A. Morphology of MDM after CAP treatment. Macrophages in the CAP-treated area were imaged 1 h and 24 h after treatment (45 sec at 4.5 kV). B. Cell viability was assessed by MTT assay. MTT conversion to formazan was measured at OD₅₇₀.</p

CAP effects on HIV-1 replication.

<p>A. Monocyte-derived macrophages treated with CAP or helium (control) were infected with HIV-1 ADA and viral replication was monitored for 15 days by RT activity in the culture supernatant. B. Results (mean±SD) are presented for RT analysis on day 15 after infection (performed as in panel A) for 7 different donors. ****p<0.0001 by Student’s unpaired two-tail <i>t</i>-test. C. MDM infected with HIV-1 ADA as in A were analyzed 4 h post infection by qPCR for positive-strand cDNA. Cells treated with AZT (3 μM) and uninfected cells are shown as negative controls. Results are presented as mean±SD for four independent infections with cells from one representative donor. ***p = 0.0004 by Student’s unpaired two-tail <i>t</i>-test. D. HIV-infected MDM were analyzed 48 h post-infection by Alu-GAG qPCR for integrated proviral DNA. Results are presented as mean±SD for four independent infections with cells from one representative donor. *p = 0.0494 by Student’s unpaired two-tail <i>t</i>-test. E. HIV-1 ADA was treated with CAP or helium (control) and used to infect MDM. Virus infection was assessed by measuring reverse transcriptase activity in culture supernatant on day 10–15 post-infection. Results (mean±SD) are presented for 6 experiments with MDM from independent donors. ***p<0.001 by Student’s unpaired two-tail <i>t</i>-test.</p

Supplemental Figure 1. Primary antibody and GFP vector controls do not localize to L. monocytogenes membrane protrusions or invaginations.

(A) HeLa cells were infected with wild-type L. monocytogenes for 6 hours. Samples were fixed then stained with normal mouse IgG antibody (green), Alexa594-phalloidin (red) to visualize F-actin and DAPI (blue) to visualize DNA. The control IgG is not enriched at the plasma membrane surrounding membrane protrusions (top) or at invaginations (bottom). Open arrowheads indicate L. monocytogenes bacteria (blue) while solid arrowheads point to protruding bacteria (red). Scale bar is 5 μm. (B) HeLa cells transfected with GFP alone (green) were infected with wild-type L. monocytogenes for 6 hours. They were then fixed and stained with Alexa594-phalloidin (red) to visualize F-actin and DAPI (blue) to visualize DNA. GFP alone is not enriched at the plasma membrane surrounding the membrane protrusions (solid arrowhead) or at invaginations (open arrowhead). Scale bar is 5 μm

Supplemental Figure 5. L. monocytogenes membrane protrusions are collapsed and/or contorted in SKOV3 and A549 cell lines depleted of CD147.

<p>SKOV3 and A549 cells were treated with control non-targeting (A) or CD147 targeting siRNA (B), infected with wild-type <i>L. monocytogenes</i> for 6 hours, fixed, then stained with mouse monoclonal CD147 targeting antibodies (green), DAPI (blue) to visualize bacteria (open arrowheads) and Alexa594-phalloidin (red) to visualize F-actin and membrane protrusions. CD147 is present at the plasma membrane of normal protrusions (solid arrowheads) extending from control siRNA treated cells (A). Membrane protrusions (solid arrowheads) are collapsed and/or contorted in CD147 depleted cells (B). Scale bars are 5 μm. </p

Supplemental Figure 3. CD147 is not recruited by L. monocytogenes bacteria that are unable to induce F-actin polymerization.

<p>HeLa cells were infected with Δ<i>actA L. monocytogenes</i> for 6 hours, fixed then stained with mouse monoclonal CD147 targeting antibodies (green), DAPI (blue) to visualize bacteria and Alexa594-phalloidin (red) to visualize F-actin. CD147 is absent from the surface of intracellular bacteria (solid arrowhead). Scale bar is 10 μm.</p

Supplemental Figure 6. Confirmation of CD147 depletion in cells during L. monocytogenes cell-to-cell spreading experiments.

The same image displayed as in Figure 3A but showing that CD147 is highly depleted in L. monocytogenes infected cells treated with CD147-targeting (bottom) siRNA sequences compared to infected cells treated with non-targeting control siRNA sequences (top). Scale bar is 20 μm

Supplemental Figure 4. In cells depleted of CD147, collapsed L. monocytogenes membrane protrusions still contain ezrin while comet/rocket tails remain morphologically normal.

<p>(A) HeLa cells were treated with non-targeting control (Ctrl) or CD147-targeted (knockdown [KD]) siRNA sequences, and whole-cell lysates were collected and probed for endogenous CD147 using mouse monoclonal CD147-targeting antibodies. Loading control with α-tubulin is shown below.</p> <p>(B) Ezrin immunolocalization (green) at actin-rich <i>L. monocytogenes</i> membrane protrusions (red) generated in cells treated with non-targeting control (top) or CD147-targeting (bottom) siRNA sequences. Ezrin is present within the core of wild-type as well as collapsed <i>L. monocytogenes</i> membrane protrusions. Open arrowheads indicate the location of <i>L. monocytogenes</i> bacteria (blue) within their respective membrane protrusion. Scale bar is 10 μm.</p> <p>(C) Cytosolic comet/rocket tails (solid arrowheads) appear morphologically normal in cells treated with CD147-targeting siRNA sequences. Open arrowheads indicate collapsed and/or contorted <i>L. monocytogenes </i>membrane protrusions. Scale bar is 10 μm.</p

Vpr associates with proteasome in fission yeast and mammalian cells.

<p><b>A</b>. Vpr is displaced from the nuclear membrane by overproduction of Uch2. Fission yeast cells carrying or not carrying Uch2 were stained with DAPI 17 hrs after <i>vpr</i> gene induction. Green color, GFP; Blue color, nuclear DNA. <b>B</b>. Vpr is displaced from the nuclear membrane in the <i>cut8</i> mutant. Mts4, a fission yeast homologue of mammalian S2, is a 19S proteasome-associated protein. Cut8 displays normal phenotype at the permissive 25°C, but shows mutant phenotype at non-permissive 37°C. <b>C</b>. Co-migration of Vpr with proteasome in fission yeast cells (<b>i</b>) and HeLa cells (<b>ii</b>) analyzed by glycerol gradient. Extracts from fission yeast cells expressing <i>vpr</i> were fractionated by centrifugation on a 10–40% glycerol gradient. Equal amounts of proteins from each fraction of the gradient were separated on 12% SDS-PAGE and probed with antibodies against Vpr and 19S (Mts4) subunits of the proteasome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Wilkinson2" target="_blank">[34]</a>. Lanes 1–8 indicates different fractions collected from the top (low molecular weight) to bottom of the gradient (high molecular weight). Note that not all fractions are shown here. <b>D.i</b>. Co-immunoprecipitation shows interaction of Vpr with Mts2 in yeast cells. IP was carried out with anti-HA as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Huard1" target="_blank">[59]</a>. A HA-tag alone plasmid control was used in this experiment. The recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-HA, anti-Vpr and anti-Mts2 antibodies. CL, cell lysates; IP:HA, immunoprecipitation with a HA-tagged control plasmid; IP:HA-Vpr, immunoprecipitation with a HA-Vpr carrying plasmid. <b>ii</b>. HeLa cells were transfected with HA-Vpr or HA-Kir2.1 (control). Kir2.1 is an irrelevant protein to Vpr and used here as a control. IP was carried out with anti-HA, recovered proteins were fractionated on SDS-PAGE and immunoblotted with anti-S2 (a mammalian homologue of fission yeast Mts4) and anti-S5a antibodies. <b>iii</b>. HeLa cells were co-transfected with pSG5-ZZ-β1, which codes for a proteasomal β1subunit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011371#pone.0011371-Klare1" target="_blank">[38]</a>, or control pSG5-ZZ plasmid (Ctr) together with HA-Vpr. The protein A-tagged β1 or control protein were pulled down by anti-protein A antibody, then blotted with anti-HA antibody.</p

Proposed model of Vpr-proteasome interactions.

<p>Details are in the text.</p