Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS

Abstract

Currently, the spatial distribution of human respiratory syncytial virus (hRSV) proteins and RNAs in infected cells is still under investigation, with many unanswered questions regarding the interaction of virus-induced structures and the innate immune system. Very few studies of hRSV have used subcellular imaging as a means to explore the changes in localization of retinoic-acid-inducible gene-I (RIG-I)-like receptors or the mitochondrial antiviral signaling (MAVS) protein, in response to the infection and formation of viral structures. In this investigation, we found that both RIG-I and melanoma differentiation-associated gene 5 (MDA5) colocalized with viral genomic RNA and the nucleoprotein (N) as early as 6 h postinfection (hpi). By 12 hpi, MDA5 and MAVS were observed within large viral inclusion bodies (IB). We used a proximity ligation assay (PLA) and determined that the N protein was in close proximity to MDA5 and MAVS in IBs throughout the course of the infection. Similar results were found with the transient coexpression of N and the phosphoprotein (P). Additionally, we demonstrated that the localization of MDA5 and MAVS in IBs inhibited the expression of interferon β mRNA 27-fold following Newcastle disease virus infection. From these data, we concluded that the N likely interacts with MDA5, is in close proximity to MAVS, and localizes these molecules within IBs in order to attenuate the interferon response. To our knowledge, this is the first report of a specific function for hRSV IBs and of the hRSV N protein as a modulator of the innate immune response.

Fig 1

hRSV genomic RNA colocalized with small but not large inclusion bodies (IBs). HEp-2 cells were infected with hRSV strain A2 at an MOI of 1.0 for 6, 12, or 24 h, and subsequently hRSV N was detected using immunostaining and genomic RNA detected with a live cell hybridization probe. Column 1, hRSV N localization. Column 2, localization of viral genomic RNA. Column 3, merge of the first two columns with hRSV N staining in green and genomic RNA in red. Arrow 1, large IB containing hRSV N but not genomic RNA. Arrow 2, region containing small IBs and hRSV N colocalized with genomic RNA. A higher and lower optical plane of the cell at the 24-hpi time point is shown. A crop of the higher plane is shown to emphasize detail of the viral filaments. Scale bars, 10 μm (top 4 rows) and 5 μm (row 5).

Fig 2

hRSV IBs colocalize with MAVS and RIG-I. A549 cells were infected with RSV strain A2 at an MOI of 1.0 and incubated for 6, 12, or 24 h or were mock infected. hRSV N, MAVS, and RIG-I were detected by immunofluorescence. Column 1, RSV N staining; column 2, MAVS staining; column 3, RIG-I staining. Column 4, merge with hRSV N colored green, MAVS colored red, and RIG-I colored blue. The nucleus, stained by DAPI, is colored white. Column 5, increased-magnification view of the boxes in the merged images. Below each panel is an intensity line graph showing the intensity of each channel along the line in the cropped image. Detail of filaments in a higher optical plane of the cell at the 24-hpi time point is shown in row 5. Scale bars,10 μm (column 4, rows 1 to 4) and 5 μm (column 5 and row 5). Images for the mock infections are wide-field only due to a computational artifact of deconvolving background without positive signal.

Fig 3

hRSV IBs colocalize with MDA5 and RIG-I. A549 cells were infected with hRSV strain A2 at an MOI of 1.0 and incubated for 6, 12, or 24 h or were mock infected. hRSV N, MDA5, and RIG-I were detected by immunofluorescence. Column 1, hRSV staining; column 2, MDA5 staining; column 3, RIG-I staining. Column 4, merge with hRSV N colored green, MDA5 colored red, and RIG-I colored blue. The nucleus, stained by DAPI, is colored white. Column 5, increased-magnification view of the boxes in the merge image. Below each panel is an intensity line graph showing the intensity of each channel along the line in the cropped image. Detail of filaments in a higher optical plane of the cell at the 24-hpi time point is shown in row 5. The arrows in row 5 point to an inclusion body (upper) and a viral filament (lower). Scale bars, 10 μm (column 4, rows 1 to 4) and 5 μm (column 5 and row 5). Images for the mock infections are wide-field only due to a computational artifact of deconvolving background without positive signal.

Fig 4

MDA5 colocalized with isolated viral filaments. HEp-2 cells were infected with hRSV strain A2 at an MOI of 0.1, and viral filaments were isolated 4 dpi. Viral filaments were isolated by filtration and adsorbed onto cover glass for immunostaining. Column 1, host protein RIG-I, MDA5, or MAVS, red in the merge. Column 2, hRSV N staining, green in the merge. Column 3, hRSV F staining, blue in the merge. Column 4, merge. Column 5, intensity profile through the lines in the merged images; arrow, pixel number 1. Scale bar, 1 μm.

Fig 5

IBs formed by cDNA-based expression of hRSV N and P proteins colocalize with RIG-I, MDA5, and MAVS. Vero cells were transfected with plasmids encoding hRSV N-GFP fusion and hRSV P proteins. Cells were immunostained for N and either RIG-I, MDA5, or MAVS. Column 1, IBs marked by hRSV N-GFP and immunostaining against the N protein. Column 2, immunostaining against RIG-I (row 1), MDA5 (row 2), or MAVS (row 3). Column 3, merge of column 1 (green) with column 2 (red). Nuclei are stained with DAPI and colored blue. Column 4, a higher magnification view of the boxed region of the panels in column 3. The bar graphs show Pearson's coefficient, Manders overlap, and individual components of Manders overlap (Mx and My). Data are from five independent fields. Error bars show standard deviations. Scale bars, 10 μm (column 3) and 2.5 μm (column 4).

Fig 6

Secondary antibodies do not cross-react with the primary antibodies, nor do the fluorophors bleed through to adjacent fluorescent channels. A549 cells were infected with hRSV strain A2 at an MOI of 1.0 for 24 h and were subjected to immunostaining using a single primary antibody against hRSV N, MDA5, MAVS, or RIG-I. All three secondary antibodies against mouse (Ms), rabbit (Rb), and goat (Gt) were applied. The columns show the individual channels corresponding to the 488-, Cy3-, and Cy5-labeled antibodies and the merge. Scale bars, 10 μm.

Fig 7

hRSV N interacts with MDA5 and MAVS. Vero cells were infected with hRSV strain A2 at an MOI of 1.0 and incubated for 6 or 24 h. Cells were stained for hRSV P and were assayed for the interaction between hRSV N and either MDA5 or MAVS by PLA. Column 1, immunostaining for hRSV P. Column 2, PLA signal from either hRSV N-MDA5 assays (rows 1 and 2) or hRSV N-MAVS assays (rows 3 and 4). Column 3, merge of column 1 (green) and column 2 (red), with the nucleus stained with DAPI in blue. Column 4, magnification of the boxed region in the panels in column 3. The bar graph shows the mean number of PLA spots per cell detected for each experimental condition along with those for the negative controls (Fig. 8). Error bars are standard deviations. Scale bars, 10 μm (column 3) and 5 μm (column 4).

Fig 8

PLA gave no signal in primary antibody control cells infected with RSV. Vero cells were infected with hRSV strain A2 at an MOI of 1.0 and incubated for 6 or 24 h. Cells were stained for RSV P (green) and were assayed for the interaction between RSV N and either MDA5 or MAVS by PLA (red) without the primary antibody for hRSV N. The nucleus, stained by DAPI, is colored blue. Scale bars, 10 μm.

Fig 9

hRSV N is in proximity to MDA5 and MAVS but not RIG-I in the absence of viral infection. Vero cells were transfected with plasmids encoding either hRSV N-GFP or hRSV N-GFP and hRSV P and assayed for the interaction between hRSV N and RIG-I, MDA5, or MAVS. Column 1, GFP signal from the hRSV N-GFP. Column 2, PLA signal for hRSV N with RIG-I (rows 1 and 4), MAVS (rows 2 and 5), or hRSV N with MDA5 (rows 3 and 6). Column 3, merge of column 1 (green) with column 2 (red), with the nucleus stained with DAPI in blue. Column 4, magnification of the boxed region in the panels in column 3. Scale bars, 10 μm (column 3) and 5 μm (column 4).

Fig 10

PLA gave no signal in primary antibody control cells transfected with plasmids encoding RSV N-GFP and RSV P, RSV N-GFP alone, RSV P-GFP and RSV N, or RSV P-GFP alone. Vero cells were transfected with plasmids encoding hRSV N-GFP, hRSV N-GFP and hRSV P, hRSV P-GFP, or hRSV P-GFP and hRSV N and assayed for the interaction between hRSV N and MAVS (column 1), MDA5 (column 2), or RIG-I (column 3) without the primary antibody for hRSV N. Row 1, cells transfected with plasmids encoding hRSV N-GFP and hRSV P. Row 2, cells transfected with a plasmid encoding hRSV N-GFP only. Row 3, cells transfected with plasmids encoding hRSV P-GFP and hRSV N. Row 4, cells transfected with a plasmid encoding hRSV P-GFP only. The nucleus, stained by DAPI, is colored blue. Scale bars, 10 μm. Images are single planes of wide-field, undeconvolved Z-stacks.

Fig 11

Mitochondria are found adjacent to hRSV IBs. Vero cells were transfected with plasmids encoding hRSV N-GFP and RSV P. At 24 h posttransfection, mitochondria were labeled with Mitotracker deep red 633 and imaged live. (A) Single cell. (B) Magnification of the boxed region in panel A. (C) Three-dimensional isosurface view of a large IB from panel B surrounded by mitochondria. hRSV N-GFP is colored green, Mitotracker is colored in red, and nuclei stained by DAPI are in blue. Scale bar, 10 μm (A) and 5 μm (B).

Fig 12

hRSV N coprecipitates with GFP-MDA5. HeLa cells were transfected with plasmids encoding GFP-MDA5 and hRSV N or GFP-MAVS and hRSV N. At 24 h posttransfection, cells were lysed and subjected to IP using an anti-GFP antibody or control antibody. Precipitates were subjected to electrophoresis and Western blotting using an anti-GFP to detect the GFP-MDA5 and GFP-MAVS and an anti-hRSV N antibody to detect the hRSV N. Blots were linearly contrast enhanced.

Fig 13

hRSV P is in proximity to MDA5 and MAVS in the absence of viral infection. Vero cells were transfected with plasmids encoding either hRSV P-GFP or hRSV P-GFP and hRSV N and assayed for the interaction between hRSV P and MDA5 or MAVS. Column 1, GFP signal from the hRSV P-GFP. Column 2, PLA signal for hRSV P with MAVS (rows 1 and 3) or hRSV N with MDA5 (rows 2 and 4). Column 3, merge of column 1 (green) with column 2 (red) with the nucleus stained with DAPI in blue. Column 4, magnification of the boxed region in the panels in column 3. Scale bars, 10 μm (column 3) and 5 μm (column 4).

Fig 14

PLA produces a signal in the presence of interacting antigens but not in diffusely localized, noninteracting antigens. Vero cells were transfected with plasmids encoding either hRSV N and free GFP (N-OPT + GFP) or hRSV N-GFP alone (N-GFP). A PLA was performed against the GFP and hRSV N domains in both cases. PLA signal was quantified on a per-cell basis and summarized. Nuclei were stained with DAPI. ***, P ≤ 0.001. N.D., not detected. GFP signal is shown in green, PLA signal is shown in red, and DAPI signal is shown in blue. Scale bars, 10 μm. Error bars are standard deviations.

Fig 15

hRSV N expressed alone or with hRSV P functions to diminish interferon expression in response to infection. HeLa cells were either untransfected (control), mock transfected with Lipofectamine 2000 (LP), or transfected with 2 μg of a GFP-expressing plasmid (GFP), an hRSV N expressing plasmid (N), an hRSV P expressing plasmid (P), or both an hRSV P expressing plasmid and an hRSV N expressing plasmid (NP). After 24 h, cells were left uninfected or were infected with NDV LaSota strain (+ NDV). At 24 h postinfection, cells were harvested for RNA and qRT-PCR was performed for IFNB1 using ACTB as a loading control. (A) Experimental and control cases. (B) Selected data from panel A rescaled, along with hRSV P and a titration of the hRSV N plasmid of one-quarter the amount of DNA (0.25) and one-tenth the amount of DNA (0.1). Error bars are minimum and maximum relative quantification (RQ).

Fig 16

Immunofluorescence staining of expressed IBs yields an open ring-shaped structure. Vero cells were transfected with plasmids expressing hRSV N-GFP fusion and hRSV P proteins and immunostained for hRSV N. The nucleus was stained by DAPI. The merge panel shows the overlay of RSV N immunostaining (red), N-GFP (green), and the nucleus (blue). The image is an XY plane through the center of the largest inclusion bodies. Scale bars, 10 μm.