p38 and OGT Sequestration into Viral Inclusion Bodies in Cells Infected with Human Respiratory Syncytial Virus Suppresses MK2 Activities and Stress Granule Assembly
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Fig 1Western blot analysis of the intracellular expression of p38α, p38-P, and RSV proteins in response to RSV infection and treatment with inhibitor CMPD-1. Monolayer cultures of A549 cells were infected with recombinant wild-type RSV (wt-rRSV) at an MOI of 0.5 or were mock infected. Replicate cultures were treated with the inhibitor CMPD-1 or mock treated, beginning 30 min before infection (−30 min) or 75 min postinfection (+75 min). The cultures were harvested 18 h postinfection and analyzed by Western blotting with antibodies against p38α, p38-P, and purified RSV (which reacted here with the N, P, and SH proteins) and β-actin as a loading control. The antibodies to p38α and p38-P did not cross-react, and different exposure times were used (1 min for p38α, 10 min for p38-P, 20 s for RSV, and 3 s for actin), and thus these results do not indicate the relative abundance of p38α versus p38-P.
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Fig 2Effect of CMPD-1 treatment on RSV replication and RNA synthesis. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with the inhibitor CMPD-1 or mock treated beginning 75 min postinfection and were incubated for a total of 18 h. (A) RSV replication. The cell culture medium, from two replicate cultures per time point, was collected at 12 h, 18 h, and 24 h postinfection, and viral titers were determined by plaque assay. (B) FISH images of RSV-infected cells. Cultures were fixed and hybridized with a set of RNA probes specific to the N, P, M2-1, NS1, NS2, and F genes (red), and nuclei were stained with DAPI (blue). The samples were analyzed by confocal microscopy.
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Fig 3Immunofluorescence images of the intracellular distribution of p38-P and the viral M2-1 protein in response to RSV infection and treatment with inhibitor CMPD-1. Monolayers of A549 cell were infected with wt-rRSV at an MOI of 0.5 or were mock infected. The cells were treated with the inhibitor CMPD-1 or mock treated 75 min after infection, incubated for a total of 18 h, fixed, and immunostained using antibodies specific to M2-1 (green) and p38-P (red), and the nuclei were stained with DAPI (blue). The white arrowheads indicate examples of IBs.
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Fig 4Immunofluorescence imaging and intensity profiles of viral IBs. A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. At 18 h postinfection, the cells were fixed and stained with antibodies for either the viral M2-1 (A, green) or P (B, green) protein and p38-P (red). Nuclei were stained with DAPI (blue). The samples were analyzed by confocal microscopy. In panels A and B, the bottom row of images shows enlargements from the middle row as well as cross-sections in the x and y dimensions. The panels on the right show a single mid-cell layer from a z-stack of confocal images (top images). The white line with arrowheads below each end indicates the track of a line intensity profile across one (A) or two (B) selected IBs (bottom images).
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Fig 5Western blot analysis of the intracellular expression of MK2 and phosphorylated MK2 (MK2-P) in response to RSV infection and treatment with inhibitor CMPD-1. (A) Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Replicate cultures were treated with the inhibitor CMPD-1 or mock treated, beginning 30 min before infection (−30 min) or 75 min postinfection (+75 min). Mock-infected cells were treated with arsenite for 30 min at 18 h postinfection as a control. The cultures were harvested and analyzed by Western blotting with antibodies against MK2/MK2-P and β-actin as a loading control. (B) A549 cell cultures were infected or mock infected as for panel A and were treated with arsenite or mock treated for 30 min at 18 h postinfection. The cells were harvested and analyzed by Western blotting with antibodies against MK2/MK2-P, and β-actin was used as a loading control.
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Fig 6Immunofluorescence analysis of the intracellular distribution of p38-P and viral P (A) or M2-1 (B) protein in response to RSV infection and treatment with arsenite. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with arsenite or mock treated for 30 min at 18 h postinfection, fixed, and immunostained using antibodies to the P (A, green) or M2-1 (B, green) protein and p38-P (red). Nuclei were stained with DAPI (blue). Samples were analyzed by confocal microscopy.
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Fig 7Immunofluorescence analysis of the intracellular distribution of OGT, the viral P protein, and the SG marker TIA-1 in response to RSV infection and treatment with arsenite. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with arsenite or mock treated for 30 min at 18 h postinfection, fixed, and immunostained using antibodies specific to the P protein (green), OGT (red), and TIA-1 (violet), and nuclei were stained with DAPI (blue). Panel B shows enlargements from the bottom row of panel A, with further enlargements rendered as three-dimensional images. White arrowheads in panel B indicate representative IBs, illustrating that the IBs contain P and OGT but do not localize with TIA-1. An animation of panel B is shown in Video SA in the supplemental material.
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Fig 8Immunofluorescence analysis of the intracellular distribution of OGT, the viral M2-1 protein, and the SG marker TIA-1 in response to RSV infection and treatment with arsenite. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with arsenite or mock treated for 30 min at 18 h postinfection, fixed, and immunostained using antibodies specific to M2-1 (green), OGT (red), and TIA-1 (violet), and nuclei were stained with DAPI (blue). Panel B shows enlargements from the bottom row of panel A, with further enlargements rendered as three-dimensional images. The white arrowhead in panel B indicates a representative IB containing M2-1 and OGT, but not TIA-1, and the white arrows in panel B indicate representative SGs containing M2-1 and TIA-1. An animation of panel B is shown in Video SB in the supplemental material.
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Fig 9Immunofluorescence analysis of the intracellular distribution of OGT and OGN in response to RSV infection and treatment with arsenite. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with arsenite or mock treated for 30 min at 18 h postinfection, fixed, and immunostained using antibodies against RSV (green), OGT (red), and OGN (violet). Nuclei were stained with DAPI (blue). Panel B shows enlargements from the bottom row of panel A, with further enlargements rendered as three-dimensional images. Incidentally, the anti-RSV antibodies used in this particular experiment had been raised against whole virus and RSV staining was not limited to IBs; thus, colocalization of viral protein with OGT in IBs (one example is indicated by a white arrowhead in panel B) was much less evident than in other experiments.
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Fig 10Immunofluorescence analysis of the intracellular distribution of SGs and OGN in response to RSV infection and treatment with arsenite. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. Cultures were treated with arsenite or mock treated for 30 min at 18 h postinfection, fixed, and immunostained using antibodies against RSV (green), the SG marker eIF3 (red), and OGN (violet). Nuclei were stained with DAPI (blue). Panel B shows enlargements from the bottom row of panel A, with further enlargements rendered as three-dimensional images. White arrows in panel B show examples of colocalization of eIF3 and OGN, indicating that both are in SGs.
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Fig 11Examples of RSV-infected cells that contain IBs alone or IBs and SGs. Monolayer cultures of A549 cells were infected with wt-rRSV at an MOI of 0.5 or were mock infected. At 18 h postinfection, cells were fixed and immunostained using antibodies specific for M2-1 (green), OGT (red), and TIA-1 (violet). Nuclei were stained with DAPI (blue). White arrowheads indicate an example of a well-developed IB; white arrows indicate areas of SGs. The cell labeled “a” is an example of an RSV-infected cell that contains well-developed IBs and lacks detectable SGs; this pattern was observed in ∼95% of RSV-infected cells. The cell labeled “b” is an example of an RSV-infected cell in which the IBs are smaller and SGs also are present; this pattern was observed in ∼5% of RSV-infected cells.
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