Figure 1.
Signaling in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ, assayed by VSV-GFP plaque formation.
Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 5 TCID50/cell. After 48 h, cells were mock-treated or treated with 100 or 1000 IU/ml of the indicated IFN for 24 h. Subsequently, cells were infected with GFP-expressing VSV, and VSV plaques were enumerated 48 h later. Inhibition of VSV plaque formation is an indication of IFN signaling to create an antiviral state. The relative plaque numbers are shown, as percent of the number of plaques that formed in non-IFN-treated wells. WT and F170S HPIV1-infected cells stimulated with 100 or 1000 IU of any of the three IFNs differed significantly in their ability to restrict VSV plaque formation (P values for two-tailed T-tests are indicated).
Figure 2.
Western blot of total and phosphorylated Stat1 and Stat2 in WT or F170S HPIV1-infected Vero cells following treatment with IFN-α, -β, or -γ.
Vero cells were mock-infected or infected with WT HPIV1, F170S HPIV1, or HPIV2 at an MOI of 5 TCID50/cell. After 48 h, cells were mock-treated or treated for 30 min with 1000 IU/ml of the indicated IFN. A) Western blots were probed for total or phosphorylated (p)Stat1 and Stat2, as well as for the HPIV1 C protein and HPIV2 P protein. Alpha-tubulin was used as loading control. B) Extended exposure (over night) of the top panel in Figure 2A [“pStat1 (Tyr701)”], showing that a low level of pStat1 is detected in cells infected with WT HPIV1 in the absence of IFN treatment.
Figure 3.
Intracellular localization of Stat1 in WT or F170S HPIV1-infected Vero cells following IFN treatment.
Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID50/cell, and 48 h later were mock-treated (-IFN) or treated (+IFN) with 1000 IU/ml of IFN-β for 1 h. Cells were fixed, permeabilized, immunostained with antibodies for HPIV1 surface proteins (green) and Stat1 (red), stained with DAPI to visualize nuclei (blue), and analyzed by confocal microscopy. Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 82% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure 4.
Intracellular localization of Stat2 in WT or F170S HPIV1-infected Vero cells following IFN treatment.
Cells were infected and analyzed as described in the legend to Figure 3 except that the antibodies against Stat1 were replaced with antibodies against Stat2 (red). Representative fields are shown. Overall, 2% of the WT HPIV1-infected cells and 100% of the F170S HPIV1-infected cells showed nuclear Stat1 following IFN-β treatment.
Figure 5.
Co-immunoprecipitation of WT HPIV1 C protein and Stat1.
293 T cells were transfected with pcDNA3.1(+) plasmids expressing myc-tagged C′WT or C′F170S protein, or untagged CAT as a negative control. After 48 h, cells were mock-treated (IFN−) or treated (IFN+) with 1000 IU/ml of IFN-β for 30 min. Cell lysates were subjected to immunoprecipitation with anti-myc antibodies. Whole cell lysates and precipitates were separated on SDS-PAGE gels and analyzed by Western blot with antibodies against pStat1, Stat1, or the C protein, as indicated at the left. The experiment was carried out three times with comparable outcomes.
Figure 6.
Co-localization of Stat1 and HPIV1 C proteins in Vero cells.
Vero cells were mock-infected or infected with WT or F170S HPIV1 at an MOI of 1 TCID50/cell. After 48 h, cells were mock-treated (−IFN) or treated with 1000 IU/ml of IFN-β (+IFN) for 30 min. Cells were subsequently fixed, permeabilized, and stained for HPIV1 C proteins (red) and endogenous Stat1 protein (green). Z-stacks of Figure 6 are shown in the Videos S1, S2, S3, S4.
Figure 7.
Co-localization of C proteins and mannose6-phosphate receptor in Vero cells.
Vero cells were treated as described for Figure 6. Cells were stained for HPIV1 C proteins (red) and M6PR (green). Z-stacks of Figure 7 are shown in the Videos S5, S6, S7, S8.
Figure 8.
Co-localization of Stat1 and mannose6-phosphate receptor in Vero cells.
Vero cells were treated as described for Figure 6. Cells were stained for Stat1 (red) and M6PR (green). Z-stacks of Figure 8 are shown in the Videos S9, S10, S11, S12.
Figure 9.
Co-localization of Stat2 and mannose6-phosphate receptor in Vero cells.
Vero cells were treated as described for Figure 6. Cells were stained with for Stat2 (red) and M6PR (green). Z-stacks of Figure 9 are shown in the Videos S13, S14, S15, S16.