Respiratory Syncytial Virus Sequesters NF-κB Subunit p65 to Cytoplasmic Inclusion Bodies To Inhibit Innate Immune Signaling

Abstract

Viruses routinely employ strategies to prevent the activation of innate immune signaling in infected cells. Respiratory syncytial virus (RSV) is no exception, as it encodes two accessory proteins (NS1 and NS2) which are well established to block interferon signaling. However, RSV-encoded mechanisms for inhibiting NF-κB signaling are less well characterized. In this study, we identified RSV-mediated antagonism of this pathway, independent of the NS1 and NS2 proteins and indeed distinct from other known viral mechanisms of NF-κB inhibition. In both human and bovine RSV-infected cells, we demonstrated that the p65 subunit of NF-κB is rerouted to perinuclear puncta in the cytoplasm, which are synonymous with viral inclusion bodies (IBs), the site for viral RNA replication. Captured p65 was unable to translocate to the nucleus or transactivate a NF-κB reporter following tumor necrosis factor alpha (TNF-α) stimulation, confirming the immune-antagonistic nature of this sequestration. Subsequently, we used correlative light electron microscopy (CLEM) to colocalize the RSV N protein and p65 within bovine RSV (bRSV) IBs, which are granular, membraneless regions of cytoplasm with liquid organelle-like properties. Additional characterization of bRSV IBs indicated that although they are likely formed by liquid-liquid phase separation (LLPS), they have a differential sensitivity to hypotonic shock proportional to their size. Together, these data identify a novel mechanism for viral antagonism of innate immune signaling which relies on sequestration of the NF-κB subunit p65 to a biomolecular condensate-a mechanism conserved across the Orthopneumovirus genus and not host-cell specific. More generally, they provide additional evidence that RNA virus IBs are important immunomodulatory complexes within infected cells.IMPORTANCE Many viruses replicate almost entirely in the cytoplasm of infected cells; however, how these pathogens are able to compartmentalize their life cycle to provide favorable conditions for replication and to avoid the litany of antiviral detection mechanisms in the cytoplasm remains relatively uncharacterized. In this manuscript, we show that bovine respiratory syncytial virus (bRSV), which infects cattle, does this by generating inclusion bodies in the cytoplasm of infected cells. We confirm that both bRSV and human RSV viral RNA replication takes place in these inclusion bodies, likely meaning these organelles are a functionally conserved feature of this group of viruses (the orthopneumoviruses). Importantly, we also showed that these organelles are able to capture important innate immune transcription factors (in this case NF-KB), blocking the normal signaling processes that tell the nucleus the cell is infected, which may help us to understand how these viruses cause disease.

Keywords: LLPS; NF-κB; RSV; inclusion bodies; innate immunity; orthopneumovirus; respiratory syncytial virus; virology.

FIG 1

bRSV infection induces IRF3, but not NF-κB, nuclear translocation. (A) Vero cells uninfected (mock) or infected with bRSV at an MOI of 1 for 24 h were left untreated, stimulated with 20 ng/ml hTNF-α for 30 min, or transfected with 2.5 μg/ml poly(I:C) and incubated for 6 h at 37°C. Cells were then fixed and immunostained with anti-RSV F (green) and anti-NF-κB p65 or anti-IRF3 (red) antibodies. Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. The boxed areas are shown magnified in the panels below (inset zoom). Graphs show fluorescent line intensity profiles along the respective white lines within these inset zooms. (B) NF-κB activation in bRSV-infected MDBK cells. MDBK cells mock infected or infected with bRSV at an MOI of 1 for 24 h were left untreated or stimulated with 20 ng/ml hTNF-α for 30 min. Cells were then fixed and immunostained with anti-RSV F (green) or anti-NF-κB p65 (red) antibodies. Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. (C) 293T cells were mock infected or infected with bRSV at an MOI of 1. At 6 h p.i., cells were transfected with 100-ng NF-κB FLuc reporter and 10-ng TK-renilla luciferase and incubated at 37°C. At 18 h posttreatment (p.t.), cells were left untreated or stimulated for 16 h with 20 ng/ml hTNF-α. Cells were then lysed and analyzed for firefly and renilla luciferase activities. Graph depicts means ± SD of three replicates from the same experiment. As controls, the levels of RSV F and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by Western blotting on a fourth replicate. Statistical significance determined by ANOVA as described in the Materials and Methods; ****, P < 0.0001. (D) Vero cells mock infected or infected with bRSV at an MOI of 2 for 24 h were left untreated or stimulated with 20 ng/ml hTNF-α for 10 min. Cells were then lysed and analyzed by Western blotting for phosphorylation of p65 using phospho-specific forms of the antibody, total p65, IκBα, and RSV F. GAPDH was detected as a loading control.

FIG 2

BRSV replication induces the recruitment of the NF-κB subunit p65 into intracytoplasmic bodies distinct from stress granules. (A) MDBK cells were mock infected or infected with bRSV. At the indicated times p.i. cells were fixed and immunostained with anti-RSV F (green) and anti-NF-κB p65 (red) antibodies. Nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. (B and C) Quantification of p65 puncta in A obtained using the quantify tool of Leica LAS AF Lite software as described in the Materials and Methods. (B) Surface area of 13 p65 puncta per time point and mean area are indicated. Statistical significance determined by ANOVA as described in the Materials and Methods; n.s, nonsignificant; *, P < 0.05; ***, P < 0.001. (C) Graph showing the line intensity profiles along chosen 15-μm lines of interest (example micrographs: 15 μm drawn across a puncta or across the cytoplasm in mock cells) of an average of five puncta per time point. (D) Vero cells were infected with bRSV or mock infected. At 24 h p.i., cells were treated with 500 μM sodium arsenite or mock treated for 1 h. Cells were then fixed and immunostained with anti-G3BP1 (green) and anti-NF-κB p65 (red) antibodies. Nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope.

FIG 3

The NF-κB subunit p65 colocalizes with viral inclusion bodies independently of RSV-encoded immunomodulators. (A) Schematic depiction of the bRSV genome showing organization of the encoded genes. (B) Vero cells, mock infected or infected with bRSV for 24 h, were fixed and immunostained with rabbit anti-NF-κB p65 (red) and mouse monoclonal anti-RSV N, P, M, or F antibodies (green). Nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. Zoom panel shows magnification of IBs boxed in the merge panel. Graphs shows fluorescent intensity profiles along the indicated white lines drawn across one or two IBs. (C) MDBK cells were mock infected or infected with bRSV. At the indicated times p.i., cells were fixed and immunostained with anti-RSV N (green) and anti-NF-κB p65 (red) antibodies. Images are max intensity Z-stacks of 8 planes 0.5 μm apart. Cytoplasmic bodies (area, >0.1 μm²) from the Z-stacks were quantified in a total of 18 infected cells per time point as detailed in the Materials and Methods. (D) Number of N- and N- and p65-positive bodies per cell at the indicated time points. (E) Surface area of identified N- and N- and p65-positive IBs. Statistical significance determined by ANOVA as described in the Materials and Methods; ****, P < 0.0001. (F) Vero cells were infected with wt bRSV, ΔNS1, ΔNS2, ΔNS1ΔNS2, or ΔSH bRSV. At 24 h p.i., cells were fixed and immunostained with rabbit anti-NF-κB p65 (red) and mouse anti-RSV F (green) antibodies. Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope.

FIG 4

bRSV IBs are sites of RNA replication, but p65 does not specifically colocalize with M2-1 or nascent viral RNA in IB-associated granules (IBAGs). (A and B) MDBK cells were mock infected or infected with bRSV. After 24 h, cells were incubated with vehicle or 20 μg/ml actinomycin D (Act D) for 1 h to inhibit cellular transcription. 5-Ethynyl uridine (5EU) was then added for another 1 h and the cells fixed. 5EU incorporated into newly synthesized RNA was detected using Alexa Fluor 488-azide (green) as described in the Materials and Methods. Cells were then immunostained with anti-RSV N, M2-1, or anti-NF-κB p65 antibodies (red). Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. Bottom of A (inset zoom) shows the boxed area (in merge of bRSV, +Act D) magnified. Graphs show fluorescent intensity profiles along the indicated white lines drawn across the IBs. Asterisks in A indicate areas of increased 5EU staining within the IB. (C) Vero cells infected with bRSV for 24 h were fixed and immunostained with rabbit anti-NF-κB p65 (red) and mouse anti-M2-1 (green) antibodies. Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. Bottom image shows a higher magnification of the boxed area; scale bar corresponds to 4 μm. Graphs show fluorescent intensity profiles along the indicated white line.

FIG 5

bRSV IBs are membraneless liquid organelles. (A) High-power transmission electron microscopy (TEM) of mock- or bRSV-infected Vero cells fixed in glutaraldehyde at 24 and 48 h p.i and prepared for TEM as detailed in the Materials and Methods. N, nucleus; M, mitochondria; C, cytoplasm; IB, inclusion body; ER is indicated with black arrow. Two representative images are shown per time point. Scale bars correspond to 1 μm. (B and C) Vero cells were infected with bRSV at an MOI of 1 and incubated at 37°C for 24 h. Hypotonic shock was applied for 20 min before the cells were fixed. Confocal analysis was performed following immunostaining for bRSV N (green) and nucleus stained with DAPI (and also p65 for C). Inset zooms demonstrate the observed effects of hypotonic shock on large (i and iii) and small (ii and iv) IBs—representative images shown. (D) Correlative light electron microscopy (CLEM) of confocal microscopy immunostaining and TEM showing bRSV IBs. Vero cells infected with bRSV at an MOI of 1 were fixed at 24 or 48 h p.i., stained with antibodies against RSV N (green) and NF-κB p65 (red) and nuclei stained with DAPI. Following confocal imaging, cells were fixed in glutaraldehyde, sectioned, and visualized by TEM. Confocal (left) and TEM (middle) images of the same cells were overlaid (right) as CLEM images.

FIG 6

Coexpression of bRSV N and P proteins induces the formation of IB-like structures which can sequester p65. (A) Vero cells were cotransfected with equimolar concentrations of plasmids expressing bRSV N (pN) and/or P (pP) proteins as indicated. Following 24 h of incubation, cells were fixed and stained with anti-RSV N (green/red) and anti-RSV P (green) or anti-NF-κB p65 (red) antibodies. Bottom image shows a higher magnification of the boxed area. Graph shows fluorescent intensity profiles along the indicated white line. (B) Coimmunoprecipitation of p65. 293T cells were transfected with plasmids expressing NF-κB p65 (pP65) or empty vector (pEV) and 6 h later infected with bRSV at an MOI of 1. At 24 h p.i., cell lysates were immunoprecipitated (IP) with anti-p65 antibody or beads alone as a control. Pulldowns were analyzed by SDS-PAGE and immunoblotting (IB) using anti-p65, anti-N, or anti-P antibodies.

FIG 7

The sequestration of the NF-κB subunit p65 to cytoplasmic IBs is a conserved mechanism of orthopneumovirus immunomodulation. (A) 293T cells were mock-infected or infected with hRSV at an MOI of 1. At 6 h p.i., cells were transfected with 100-ng NF-κB FLuc reporter and 10-ng TK-renilla luciferase and incubated at 37°C. At 18 h p.t., cells were left untreated or stimulated for 16 h with 20 ng/ml hTNF-α. Cells were then lysed and analyzed for firefly and renilla luciferase activities. Graph depicts means ± SD of three replicates from the same experiment. Statistical significance determined by ANOVA as described in the Materials and Methods; ****, P < 0.0001. (B) Vero cells mock infected or infected with hRSV at an MOI of 1 for 24 h were left untreated or stimulated with 20 ng/ml hTNF-α for 30 min. Cells were then fixed and immunostained with anti-RSV F (green) or anti-NF-κB p65 (red) antibodies. Cell nuclei were stained with DAPI (blue) and images obtained using a Leica TCS SP5 confocal microscope. Graphs show line fluorescent intensity profiles along the indicated white lines. (C) MDBK, Hep2, and BEAS-2B cells were infected with b/hRSV for 24 h, fixed, and immunostained for RSV N (green) or NF-κB p65 (red). (D) Vero cells were cotransfected with equimolar concentrations of plasmids expressing hRSV N (pN) and/or P (pP) proteins as indicated. Following 24 h of incubation, cells were fixed and stained with anti-RSV N (green/red) and anti-RSV P (green) or anti-NF-κB p65 (red) antibodies. Cell nuclei were stained with DAPI (blue) and confocal analysis performed. The bottom image shows a higher magnification of the boxed area, and the graph shows the fluorescent intensity profiles along the indicated white line.