Production of chemokines by APC infected with recombinant LASV.

<p>The production of CC and CXC chemokines by DC (A, B) and MP (C, D) was assessed after mock infection (white bars), or infection with recombinant wild-type LASV (gray bars) or rLASV NP-D389A/G392A (rNP LASV) (black bars). (A, C) The synthesis of mRNAs was analyzed by RT-qPCR 24 h after infection. (B, D) The protein levels released in the supernatants were quantified by ELISA 24 h (only for CXCL9, 10 and 11) and 48 h after infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 4 (A, C) and 3–7 (B, D) independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (p<0.001).</p

Production of type I IFN and chemokine mRNA in DC and MP infected with LASV and MOPV.

<p>We obtained mRNA from DC and MP 24(black bars), MOPV (gray bars), or after mock infection (white bars). (A) The amounts of type I IFN mRNA produced by DC and MP are shown as a gene mRNA/β-actin mRNA ratio. The synthesis of chemokine mRNA was evaluated with the GAPDH gene used as a housekeeping gene, in both DC (B) and MP (C). <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± standard error (SE) of 4 and 5 independent experiments, for LASV- and MOPV-infected cells, respectively. Significant differences between mock- and virus-infected cells are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** (<i>p</i><0.001).</p

Release of chemokines into the supernatants of DC and MP infected with LASV and MOPV.

<p>The levels of CC and CXC chemokines were quantified by ELISA in the supernatants of MP (A) and DC (B) 24 and 72 h after mock (white bars), MOPV (gray bars), and LASV (black bars) infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml and ng/ml for CC and CXC chemokines, respectively. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** (<i>p</i><0.001).</p

Role of type I IFN in the production of chemokines in MOPV- and LASV-infected iDC/T-cell cocultures.

<p>(A) The synthesis of mRNAs for CCL4, CCL7, CXCL10 and CXCL11 was evaluated in mock- (gray bars) or MOPV- (black bars) infected iDC cocultured with naïve T cells for 2 days in the presence of neutralizing Ab against CD118. (B) The levels of these mRNAs then were quantified 2 days after restimulation with mock- (gray bars) or inactivated MOPV- (black bars) pulsed iDC of T cells previously cocultured with mock- or MOPV-infected iDC in the presence of irrelevant IgG2a or CD118-neutralizing Ab. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 3 independent experiments.</p

Production of chemokines by MOPV- and LASV-infected DC cocultured with T cells and correlation with type I IFN synthesis.

<p>(A) Levels of CC chemokine and CXCL11 mRNA were quantified relative to GAPDH mRNA levels, after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. Levels of CXCL10 mRNA were evaluated in iDC/T cell coculture 2 days after each stimulation of the T cells with infected or inactivated virus-stimulated iDC. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± standard error (SE) of 4 independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05) and ** (<i>p</i><0.01). (B) The levels of CC and CXC chemokines were quantified by ELISA in the supernatants after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml, except for CXCL10 expressed in ng/ml. Significant differences are indicated as follows: * (<i>p</i><0.05). (C) Correlation between CCL4, CCL7, CXCL10, and CXCL11 mRNA levels and type I IFN synthesis by MOPV- or LASV-infected iDC cultured with naïve T cells 2 days after infection, represented by a linear regression with a correlation coefficient <i>r</i> and a probability of correlation <i>p</i>.</p

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</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

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

Genetic elements swapping indicates the mode of virion assembly as a dominant determinant in the virus susceptibility to IFITMs.

<p>Glycoproteins of IFITM-resistant viruses were used to pseudotype viral cores of IFITM-susceptible viruses (as indicated). HIV-1 virions presenting the HCV E1/E2 glycoproteins were produced in the presence of IFITMs by co-transfection of the respective DNAs (1:1 ratio). VSV pseudoparticles (VSVpp) incorporating the RVFV GnGc in the presence of IFITMs were produced in HEK293T transfected with DNAs coding for GnGc along with IFITMs (0.7:1 ratio), following challenge with a viral stock of ΔG-VSV virus that had been previously complemented with the G protein to allow its entry into cells. After entry, absence of G allows the production of novel virion particles that assembled in the presence of IFITMs and of the RVFV GnGc glycoprotein. Residual input virus was neutralized by incubation with an anti-G neutralizing antibody. Virion particles were purified from the supernatant of virus-producing cells, normalized and used to challenge target cells in a classical single round of infection prior to flow cytometry analysis. The panels present typical results, while the graphs present averages and SEM of 3 to 5 different experiments. *, p≤0.05 after a Student t test.</p

Silencing of endogenous IFITMs increases the infectivity of virions of different viruses and increases EBOV spread in primary macrophages.

<p>A) Endogenous IFITM1, 2 and 3 were silenced by shRNA-mediated lentiviral transduction along with shRNA-control silenced cells and then treated according to the scheme provided. B) Expression and extracellular release properties of endogenously-expressed IFITMs in the uninfected cell types used here (cells and sup., respectively). The basal expression levels of IFITM proteins were measured in the cell types mentioned above with a pool of anti-IFITM1, 2 and 3 antibodies in cell lysates and supernatants purified by ultracentrifugation through sucrose, as viral particles in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.g002" target="_blank">Fig 2</a>. The same amounts of cells used elsewhere for viral production and in the case of primary cells, the same donors were used. C) In the case of primary cells, primary blood lymphocytes were stimulated with 1 μg/ml PHA and 150 U/ml of Interleukin 2 (IL2) for twenty-four hours, then challenged with HIV-1 viral vectors expressing either control (Luciferase), or IFITMs-specific shRNAs and enriched in knockdown cells following a three-day selection in Puromycin (resistance coded by the shRNA vector). Kd-cells were then challenged with the indicated viruses at MOI comprised between 0.1 and 0.5 to obtain virus-producing cells, prior to extensive cell washing to remove input virus. Newly produced virion particles were collected 1 to 2 days afterwards (5 days for HIV-1), purified and normalized prior to WB and infectivity analyses. The infectivity of virions purified from kd-cells was measured on HeLaP4 (HeLa cells bearing the HIV-1 receptors and an LTR-driven promoter driving β-galactosidase expression, used for HIV-1 and VSV and analyzed twenty-four and sixteen hours after challenge by β-gal assay and FACS, respectively) or Vero/hSLAM (MeV, flow cytometry). Primary macrophages were challenged with shRNA-coding vectors in the presence of Vpx-containing virion-like particles (VLPs-Vpx) to increase the efficiency of silencing and then challenged with EBOV at an MOI of 0.3. Pictures of infected cultures were collected with a Leica DM IRB inverted microscope. The graphs present averages and SEM of 4 to 5 independent experiments with cells obtained from different donors. *, p≤0.05 after a Student t test.</p