Heterocellular induction of interferon by negative-sense RNA viruses

Abstract

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.

Fig. 1

At two days p.i., the uninfected cells surrounding developing plaques may (plaque 1), or may not (plaque 2), be positive for MxA. Monolayers of A549 cells grown on coverslips were infected at an moi of 0.001 pfu/cell. At 48 h p.i. the cells were fixed, stained with anti-NP and anti-MxA antibodies, and visualised using a Nikon Microphot-FXA immunofluorescence microscope.

Fig. 2

Characterisation of the A549/pr(IFN-β).GFP reporter cells. (A). Monolayers of A549 cells were either mock-infected or infected with 5 pfu per cell of a stock of MuV (ori) known to be a good inducer of IFN; at 4, 6 and 8 h p.i. the cells were trypsinised to a single cell suspension and the percentage of GFP-positive cells estimated by FACS analysis. (B). Confluent monolayers of A549/pr(IFN-β).GFP cells were grown in 60 mm dishes that contained coverslips and infected at various multiplicities of infection of MuV(ori). At 12 h p.i. the coverslips were fixed and those cells expressing GFP visualised using a Nikon Microphot-FXA fluorescence microscopy. In addition, the amount of IFN secreted into the culture medium was determined (right hand column). (C). A549/pr(IFN-β).GFP, A549/pr(IFN-β).GFP/BVBV.N^pro or A549/pr(IFN-β).GFP/HCV.NS34a cells were either mock-infected of infected with MuV(ori) at 5 pfu/cell. At 12 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. (D). A549/pr(IFN-β).GFP were, or were not (control), treated with aphidicolin (1 μg/ml) for 16 h, infected with MuV (ori) at 5 pfu/cell in the presence or absence of aphidicolin as appropriate, fixed at 16 h p.i. and GFP-positive cells visualised by fluorescence microscopy.

Fig. 3

Detection of GFP-positive cells following infection of A549/pr(IFN-β).GFP cells with a panel of negative strand RNA viruses. A549/pr(IFN-β).GFP cells were infected with MuV (cl3), PIV2, PIV3, PIV5, FLUAV or BUNV at 2–5 pfu/cell. At 16 h p.i. the cells were fixed and immunostained with an anti-NP mAb. GFP-positive and virus-infected cells were visualised by fluorescence microscopy. The presence of the nuclei in the merge images was visualised by DAPI staining. Note that MuV (cl3/30) was originally plaque-purified from MuV (ori).

Fig. 4

Detection of GFP-positive cells within developing plaques of PIV5. A549/pr(IFN-β).GFP cells grown on coverslips were infected at an moi of 0.001 pfu/cell. At 2 and 4 days p.i. the cells were fixed and immunostained with an anti-NP, and GFP-positive cells and infected cells visualised by fluorescence microscopy. Note that at two days p.i. there were a mixture of plaques which did, or did not, contain GFP-positive cells. By 4 days p.i. GFP-positive cells could be detected in all plaques, although the majority of infected cells still remained negative for GFP. The presence of the nuclei in the merge images was visualised by DAPI staining.

Fig. 5

At two days p.i. there are a mixture of PIV5 plaques in which the IFN-β promoter has, or has not, been activated in some infected cells. A549/pr(IFN-β). GFP cells grown on coverslips were mock-infected, or were infected at an moi of 0.001 pfu/cell. At 2 days p.i. the cells were fixed and immunostained with anti-NP and anti-MxA antibodies. GFP-positive cells (green), MxA-positive cells (blue) and infected cells (red) were visualised by confocal microscopy. To show that MxA is induced by IFN, its cytoplasmic distribution, and specificity of the antibody, mock-infected cells were (+ IFN) or were not (−IFN) treated with IFN for 20 h and immunostained as above. Note that expression of GFP correlated with whether the uninfected cells surrounding the plaque were positive for MxA; if a plaque contained a GFP-positive cell the surrounding cells were positive for MxA, if not they were negative.

Fig. 6

Heterocellular induction of the IFN-β promoter in developing plaques of PIV2, PIV3, FLUAV and BUNV. (A) PIV2, PIV3, FLUAV and BUNV form larger plaques on A549/BVDV-N^pro cells (that cannot to produce IFN) compared to parental A549 cells (which can produce and respond to IFN). (B) At 2 days p.i. some, but not all, PIV2, PIV3, FLUAV and BUNV plaques contain a few cells in which the IFN-β promoter has been activated. A549/pr(IFN-β).GFP cells grown on coverslips were infected at an moi of 0.001 pfu/cell. At 2 days p.i. the cells were fixed and immunostained with anti-NP and anti-MxA antibodies. GFP-positive cells (green), MxA-positive cells (blue/purple) and infected cells (red) were visualised by fluorescence microscopy. The presence of the nuclei in the merge images was visualised by DAPI staining. Note that expression of GFP always correlated with whether the uninfected cells surrounding the plaque were positive for MxA; if a plaque contained a GFP-positive cell the surrounding cells were positive for MxA, if not they were negative.