Cell-cell fusion induced by measles virus amplifies the type I interferon response

Abstract

Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-beta) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-beta response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-alpha/beta production, an amplified IFN-beta response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-beta gene deficiencies were trans complemented to induce IFN-beta production. Production of IFN-beta and IFN-alpha was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-alpha/beta. The amplification of IFN-beta production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-alpha/beta production in infected cells, and this indicates that MGC contribute to the antiviral immune response.

FIG. 1.

Role of cell-cell fusion and MeV infection in the IFN-β response. (A) Syncytium formation (scored as a minus sign [−] for no syncytium and from + to 6+, graded by integrating the numbers and sizes of live adherent syncytia plus floating dead syncytia) percentage of cells expressing the MeV N protein determined by flow cytometry, and IFN-α/β production at 7 days p.i. of 293T/CD46⁺ cells with MeV (left) or recombinant chimerical MGV (right) in the absence (dotted columns) or presence (black columns) of 10 μg/ml of FIP. (B) Syncytium formation and accumulation of MeV N (top) and IFN-β (bottom) transcripts at 23 h p.i. in 293T/CD46⁺ cells transiently expressing H^EdF (grid columns) or H^KAF (checked columns), respectively. 293T/CD46⁺ cells were infected with MGV at different MOIs, and they were then transfected at 2 h p.i. (C) Dose-response relationship of MeV F transcription and IFN-β mRNA accumulation at 30 h p.i. and IFN-α/β secretion at 3 days p.i. with the MOIs of MeV used to infect 293T/CD46⁺ cells in the absence (dotted columns) or presence (black columns) of 10 μg/ml FIP. Data are mean values ± standard deviations (SD) from two to three independent experiments. ND, not detected. † indicates cell cytoxicity.

FIG. 2.

Similar dose-dependent inhibition of cell-cell fusion and IFN-β gene transcription by FIP. 293T/CD46⁺ (A and C) or HeLa (B) cells were infected with MeV at an MOI of 1 prior to the addition of increasing amounts of FIP. (A) Micrographs of adherent cells stained with Hoechst 33342 (magnification, ×400) at 72 h p.i. Cells containing more than three nuclei were considered to be syncytia (white arrows). (B) Dose-dependent inhibition of cell-cell fusion by FIP quantified by colorimetric β-Gal reporter gene expression assay. OD, optical density. (C) Dose-dependent inhibition of IFN-β mRNA accumulation by FIP at 30 h p.i. Data are means ± SD of triplicate experiments.

FIG. 3.

MeV-induced syncytia are dynamic entities with an extended life span. MeV-infected 293T/CD46⁺ cells were cultured overnight in the presence of FIP and then cultured in the absence (A and B) or the presence (C) of FIP (10 μg/ml), with recorded imaging for the next 60 h by time lapse microscopy. Images at a magnification of ×10 (numerical aperture, 0.25) were extracted from Fig. S3A in the supplemental material and another video not shown at 12.83 h, 15 h, 16.66 h, 25.33 h, 32.5 h, and 35.33 h. (B) The duration of each stage was evaluated and expressed as means ± SD of 17 microscopic areas from three to four separate experiments. The frequency was estimated and indicated as the proportion (percent) that underwent transition through a given stage.

FIG. 4.

Reciprocal trans-complementation of RIG-I- and IFN-β-deficient cells by MeV-induced fusion. RIG-I-deficient Huh7.5 or IFN-β-deficient Vero cells were infected with MeV at an MOI of 1 and cocultured 8 h later with uninfected Vero and Huh7.5 cells (ratio, 1:1), respectively. The cocultures were treated or not treated with 10 μg/ml of FIP. Cell-free supernatants were collected at 30 and 60 h p.i. to measure IFN-α/β production. At the end of the coculture, the cell monolayers were stained for fluorescent nuclei (magnification, ×400) for counting within every syncytium indicated by arrows. Data are from one representative experiment out of two. ND, not detected.

FIG. 5.

Unlike the IFN-β gene, IRF-7 gene expression does not correlate with cell-cell fusion. 293T/CD46⁺ cells (left) and TEC (right) were either treated with 1,000 IU/ml of recombinant human IFN-β or infected with MeV at an MOI of 1 and cultured in the absence or presence of FIP (10 μg/ml). Expression of IFN-β (top dotted histograms) and IRF-7 (bottom black histograms) mRNA was quantified at 30 h p.i. Data from one representative experiment out of two or three are shown. ND, not detected.

FIG. 6.

Mature, but not immature, DC exhibit fusion-dependent amplification of IFN-α and IFN-β responses. iDC and mDC were mock infected or infected with MeV at an MOI of 0.1 in the absence (dotted columns) or the presence (black columns) of FIP (100 μg/ml). Syncytium formation was scored for each condition as described in Fig. 1. (A) CD150 expression in iDC and mDC cultures was analyzed by flow cytometry. (B) MeV F transcript accumulation in iDC and mDC cultures was measured at 3 days p.i. (C) Secreted bioactive IFN-α/β in cell-free supernatants collected at 3 days p.i. (C and D) IFN-α (D) and IFN-β (E) were measured by ELISA at 3 days p.i. (F and G) Accumulation of IFN-β (F) and IRF-7 (G) transcripts at 3 days p.i. in cDC cultures in the absence or the presence of FIP (100 μg/ml). Data are mean values from two to five separate experiments. ND, not detected; MFI, mean fluorescence intensity.

FIG. 7.

Nuclear translocation of IRF-3 can be triggered within MeV-induced syncytia. (A) Nuclear translocation of GFP-IRF-3 within syncytia of 293T/CD46⁺ cells infected by MeV at an MOI of 1. Microphotographs (magnification, ×400) show morphology (top panels), Hoechst-labeled nuclei (middle panels), and GFP-IRF-3-labeled nuclei (bottom panels) at 30 h p.i. Micrographs of uninfected cells (mock) and cells infected with MeV in the absence (MeV) or the presence (10 μg/ml) (MeV + FIP) of FIP are shown. Data are from one representative experiment out of four. (B) Three-color overlays of confocal images showing the distribution of GFP-IRF-3 (green), N (red), and nuclei (Draq5) (blue) in 293T/CD46⁺ cells infected or not infected with MeV at an MOI of 0.1 in the presence or absence of FIP and transfected with GFP-IRF-3. Syncytium images were taken at three morphological stages, flat adherent, retracting, and smooth ball, respectively. The whole set of one-color images used to build the overlays is shown in supplemental data at hal.archives-ouvertes.fr/hal-00169132. (C) Nuclear localization of endogenous IRF-3 in MGC derived from MeV-infected mDC. mDC were mock treated (left) (magnification, ×630) or infected with MeV at an MOI of 0.1 in the absence (middle) (magnification, ×400) or the presence (right) (magnification, ×630) of FIP (100 μg/ml). At 3 days p.i., mDC cultures were stained with anti-IRF-3 (green) (left) and with nucleus stain (Hoechst 33343) (blue). Cells were analyzed with an Axioplan microscope.