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

Experimental overview of the present study.

<p>Snapshot presentation of the viruses used in this study and schematic color-coded representation of the intracellular locations of virion particle assembly (A). Experimental setup used to determine effects of IFITMs on the formation of infectious viral particles (B). A comprehensive table of the viral systems used is provided in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s003" target="_blank">S1 Fig</a>.</p

IFITMs associate to viral particles of most, albeit not all, viruses tested and do not induce detectable changes in mature glycoproteins incorporation.

<p>A) Virion particles produced in presence or absence of IFITMs and their corresponding cellular lysates were analyzed by WB. Virions were harvested from the cell supernatant and purified by ultracentrifugation through sucrose, with the exception of AAV for which cells were first lysed by freeze/thaw and then virions were purified by ultracentrifugation on a four-step iodixanol gradient. Load.; loading controls using either anti-actin, -tubulin or -EF1α antibodies. Virus-specific antibodies were used according to their availability. In the case of MPMV for which antibodies were not available, CA was identified by comparison with control supernatants and quantified after <i>Coomassie</i> staining and densitometry. The panels display representative results of 3 to 5 independent experiments. B) When available, the same viral preparations displayed in A were probed with antibodies specific to the indicated mature glycoproteins. The panels present representative results. The intracellular processing pathways of the viral glycoproteins analyzed is shown schematically at the right.</p

Effect of IFITMs on the production of fully infectious viral particles.

<p>Normalized amounts of virion particles produced in the presence of absence of IFITMs were used to challenge target cells prior to analysis of their intrinsic infectivity by FACS, FFA or TCID50, according to the specific virus and as presented in Supplemental <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s003" target="_blank">S1 Fig</a>. For each virus, infectivity has been normalized to control viruses produced in the absence of IFITMs. The graph presents averages and SEM of 3 to 6 independent experiments. *, statistically significant upon a Student t test, p≤0.05.</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

Comparison between the antiviral effect of IFITMs reported in the literature for the different viruses and mediated by the pool of IFITM proteins in target cells, with the negative imprinting of the virion particles infectivity reported in this study.

<p>Given their high identity, the antiviral effects of IFITM2 and 3 are presented together, separately from those of IFITM1. Variations in the magnitude of the antiviral effects reported in the different studies have not been taken into account here, as they are likely influenced by the specific experimental conditions used, so that the effects of IFITMs on viral infectivity are presented as negative, absent (none), or controversial, even when a single conflicting report exists. When data in the literature was not directly comparable to ours (i.e. the same virus was not used), data was compared to its closest relative, marked in <i>italicus</i>. The effects of the expression of IFITMs in target cells against AAV and MeV were measured in this study and are presented in Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.s008" target="_blank">S6 Fig</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006610#ppat.1006610.g007" target="_blank">Fig 7C</a>, respectively.</p