Membrane perturbation elicits an IRF3-dependent, interferon-independent antiviral response

Abstract

We previously found that enveloped virus binding and penetration are necessary to initiate an interferon-independent, IRF3-mediated antiviral response. To investigate whether membrane perturbations that accompany membrane fusion-dependent enveloped-virus entry are necessary and sufficient for antiviral-state induction, we utilized a reovirus fusion-associated small transmembrane (FAST) protein. Membrane disturbances during FAST protein-mediated fusion, in the absence of additional innate immune response triggers, are sufficient to elicit interferon-stimulated gene induction and establishment of an antiviral state. Using sensors of membrane disruption to activate an IRF3-dependent, interferon-independent antiviral state may provide cells with a rapid, broad-spectrum innate immune response to enveloped-virus infections.

Fig. 1.

p14(wt), but not the ectodomain mutant p14Δ30, is capable of inducing pore formation and ISG 56 mRNA accumulation. (A) Schematic diagram of the important domains within the p14 proteins. The p14Δ30 mutant lacks the first 30 NH₂-terminal amino acids, including the hydrophobic patch. (B) The extent of pore formation was measured by adding calcein red-labeled Vero cells to QM5 cells cotransfected with eGFP and p14(wt) or p14Δ30 for 4 h. Pore formation was quantified by analyzing the percentage of gated eGFP-expressing cells that acquired the calcein red from the Vero cells, plotted against the forward scatter (FSC). (C) Vero cells transfected with pcDNA3.1 (EV), p14(wt), or p14Δ30 plasmid DNA were washed, trypsinized, and placed onto naïve human embryonic lung fibroblasts. RNA was harvested at 16 h post-cell transfer, followed by RT-PCR using primers specific for human ISG56 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Fig. 2.

p14(wt)-Mediated cell-cell fusion induces an antiviral state in human fibroblasts. (A) Purification of p14 proteins following ion exchange chromatography. One microgram each of p14(wt) and p14Δ30 was run on a 12% SDS-polyacrylamide gel and silver stained. For Western immunoblot analysis, 100 ng each of p14(wt) and p14Δ30 was run on a 12% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. A horseradish peroxidase-conjugated 6×His antibody was used to detect the p14 proteins. Mr, molecular mass marker. (B) QM5 cells were transfected with 4 μg of purified p14(wt) or p14Δ30 proteins for 8 h to allow fusion to proceed. Cells were fixed and Giemsa stained, and light microscopy images were captured at a magnification of ×200. Syncytia are outlined in white. (C) Following a 24-h treatment with purified p14(wt) or p14Δ30 proteins, human fibroblasts were infected with VSV expressing GFP from the viral promoter. GFP fluorescence was detected 24 h postinfection using a Typhoon Trio imager (GE Healthcare). Results from a representative experiment are presented. The level of GFP expression was quantified using ImageQuant TL software (GE Healthcare) and expressed as a percentage of fluorescence relative to that of LF2000-treated wells. Cells incubated with no VSV or VSV only were included as controls.

Fig. 3.

p14(wt) triggers an antiviral state that is dependent on IRF3. Following a 24-h treatment with 4 μg of purified p14(wt) or p14Δ30 protein, wild-type (wt) (A) and IRF3^−/− (B) MEFs were infected with VSV expressing GFP from the viral promoter. GFP fluorescence was detected 24 h postinfection using a Typhoon Trio imager (GE Healthcare). The level of GFP expression was quantified using ImageQuant TL software (GE Healthcare) and expressed as a percentage of fluorescence relative to that of LF2000-treated wells. Data are presented as means ± standard errors of the means (SEM) from three independent experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey's post hoc test, comparing all treatments to that with LF2000 alone. Cells incubated with no VSV or with VSV only were included as controls. **, P > 0.001.