The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome - PubMed (original) (raw)
The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome
Henrick Schomacker et al. PLoS One. 2012.
Abstract
Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
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.
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