M-hMPV treated-MDMs release low level of IFN-α and IFN-β.

<p>MDMs were treated with elution buffer, LPS 10 ng/mL, M-hMPV 0.172 and 0.0172 nM. Supernatants were collected after 24 h and tested for IFN-α and IFN-β production by ELISA. Bar graph represents concentrations values expressed in pg/mL. Data are represented as means of three experiments ± S.D.</p

MoDCs express the CD1a molecule while MDMs express the CD14 molecule.

<p>Monocytes were differentiated during 5 days in complete RPMI medium containing 40 ng/mL GM- CSF and 250 U/mL IL-4 for moDCs and 40 ng/mL M-CSF for MDMs. Phenotype of cells were analyzed on day 5 for the expression of CD14, CD1a and HLA-DR. Data represent histograms with isotype control (bold line) and monocyte-differentiated cells (filled profile).</p

Percentage of apoptotic M-hMPV-treated moDCs.

<p>After 24 h of stimulation, M-hMPV-treated moDCs were labeled by DiOC6(3) and propidium iodide prior analysis by flow cytometry. Data are representative of one out of two independent experiments.</p

M-hMPV activated-DCs present antigen to T cells which induce the production of IFN-γ.

<p>moDCs were treated for 24 h with LPS 10 ng/mL and M-hMPV protein 0.344, 0.172, 0.086, 0.034 nM. Cells were harvested after treatment, washed and cultured for 5 days with allogeneic purified T-cells (2×10⁵/well) at a DC/T ratio ranging between 1∶5 and 1∶40. The amount of IFN-γ in the cell-free supernatants of the co-culture was measured by ELISA. Data are represented as means of three experiments ± S.D.</p

MoDCs and MDMs treated with M-hMPV protein produce high level of cytokines and chemokines respectively.

<p>(A) Supernatants of cells treated for 24 h with elution buffer, LPS 10 ng/mL (positive control) and M-hMPV 0.172, 0.086, 0.034, 0.0172 nM were assayed for the level of IL-8, IL-6, TNF, IL-12p70, IL-1β and IL-10 cytokines by CBA. (B) Supernatant of MDMs treated with elution buffer, LPS 10 ng/mL (positive control), M-hMPV 0.172 and 0.0172 nM were assayed for MIP-1β, RANTES and TNF production by CBA. Bar graph represents concentrations values expressed in ng/mL. Data are represented as means of three experiments ± S.D.</p

M-hMPV protein binds APCs and is internalized.

<p>(A) Binding experiment. APCs were incubated with increased concentrations of M-hMPV-Fluorescein protein for 20 min at 4°C, washed and then analyzed by FACS. Data are shown as mean fluorescence intensity. (B) Competition experiment. APCs were incubated with M-hMPV-Fluorescein protein 4.1 nM for 20 min at 4°C, washed before addition of increased concentration of unlabeled M-hMPV protein for 20 min at 4°C. Results are shown as % of mean fluorescent intensity of M-hMPV-Fluorescein protein binding. (C) Internalization experiment. Cells were incubated with 1.3 nM M-hMPV-Fluorescein protein during 20 min at 4°C, washed and incubated at different time points at 37°C. Cells were then washed and analyzed. Data are shown as mean fluorescence intensity. (D) Confocal microscopy. Cells were incubated with 0.17 nM M-hMPV protein 30 min at 4°C and then washed and incubated or not at 37°C for 30 min. Observation was realized by confocal microscopy. Data represent one out of two experiments.</p

M-hMPV protein induces maturation of moDCs.

<p>moDCs were treated with M-hMPV protein 0.172 nM (gray filled) and 0.0172 nM (green line), LPS 10 ng/mL (black line), M-hMPV 0.172 nM+polymyxin (orange line), M-hMPV heat treated at 100°C–20 min (pink dotted line) or untreated (iDCs) (gray line) and the phenotype was analyzed after 24 h of treatment. Cells were stained with labeled CD80, CD83, CD86 and CD40 antibodies. Data were acquired by a FACScan and analyzed by CellQuest Pro software. Histograms represent mean fluorescence intensity. Data shown are representative of one out of five independent experiments.</p

M-hMPV bacterial recombinant protein is successfully produced.

<p>(A) SDS-PAGE analysis of M-hMPV recombinant protein was performed after purification by Ni-NTA resin. (B) M-hMPV protein was analyzed by western blot using M-hMPV 1 µg/mL monoclonal antibodies and HCV monoclonal antibody for the negative control together with species-specific secondary antibody goat anti-mouse IgG H+L labeled with alkaline phosphatase.</p

CCR5 usage relieves the negative effects of IFITM3 on HIV-1 replication and on its ability to decrease the virion particles infectivity.

<p>A) Human CCR5 was introduced in the IFITM3-stable SupT1 cells used before, by retroviral-mediated gene transduction and cells were challenged with the indicated viruses. HIV spreading was assessed by exo-RT activity over time (day 0 through 7). The panels and the histogram overlay present the patterns of expression obtained for IFITM3 and CCR5 following WB and flow cytometry analyses. The graph presents normalized data obtained in 2 to 3 independent experiments. B) Virions obtained at late times after infection were harvested, normalized and used to infect HeLaP5 cells that contain a β-galactosidase reporter gene under the control of the HIV-1 LTR.</p

CD45 depletion excludes a potentially confounding role of exosome-incorporated IFITMs on virion infectivity.

<p>SupT1 cells stably expressing the different IFITMs were infected with HIV-1 and VSV. At a late time after infection of the cell culture, supernatants containing newly-produced virions were harvested and divided in two fractions that were either incubated with CD45-conjugated microbeads or left untreated. After the microbeads removal, virion particles were purified by ultracentrifugation, normalized and then used for WB and infectivity analyses. The WB panels present typical results obtained, while the graph presents averages and SEM obtained in 3 independent experiments. No statistically significant differences were observed between depleted and non-depleted fractions, after a Student t test.</p