Article Figures & Data
Figures
- FIG. 1.
Residues 81 to 120 of NiV P are important for function in viral RNA synthesis. BSR-T7 cells were transfected with the NiV GFP-CAT minigenome plasmid along with T7 expression plasmids encoding NiV N, L, and the indicated HA-tagged NiV P constructs (wedges indicate 50, 100, and 200 ng plasmid transfected). NiV P mutant constructs include (A) internal deletions spanning residues 51 to 100 and (B) internal deletions spanning residues 101 to 150. Control samples (lanes 1 and 2) include transfection of all components of the minireplicon, with the exception of the NiV L and P plasmids, respectively. CAT expression levels, measured at 24 hpt, were normalized to the activity of constitutively expressed firefly luciferase and are shown as percentages of WT P activity (lane 3). The Western blots below the graphs indicate expression levels of P protein constructs and include β-tubulin as a loading control. IB, immunoblot.
- FIG. 2.
NiV P residues 111 to 140 are critical for efficient inhibition of IFN signaling. NiV P WT and mutant constructs were cotransfected with an IFN-inducible, ISG54-driven firefly luciferase plasmid and a Renilla luciferase plasmid into 293T cells. NiV P mutant constructs include (A) internal deletions spanning residues 51 to 100 and (B) internal deletions spanning residues 101 to 150. At 24 hpt, cells were treated with IFN-β for 16 h. Induction levels are shown as a ratios of firefly-to-Renilla luciferase levels. Western blotting with anti-HA antibody for the NiV constructs is shown below each graph. IB, immunoblot.
- FIG. 3.
NiV P, V, and W IFN signaling mutants fail to bind and inhibit STAT1. (A) 293T cells were transfected with HA-tagged NiV P WT or internal deletion constructs spanning residues 51 to 150. At 24 hpt, immunoprecipitation (IP) was performed with anti-HA antibody and Western blotting was performed with anti-STAT1 antibody. (B) 293T cells were transfected with STAT1 binding mutant forms of P as indicated and treated with 1,000 U/ml IFN-β prior to lysis. Lysates were analyzed with antiserum specific for the Y701-phosphorylated form of STAT1 and total STAT1. (C) NiV V and W deletion mutant constructs spanning residues 101 to 150 were transfected into 293T cells and immunoprecipitated as described for panel A. IB, immunoblot; WCL, whole-cell lysate.
- FIG. 4.
Separation of NiV P functional domains in minireplicon and IFN signaling assays. (A) BSR-T7 cells were transfected as described for Fig. 1, with plasmids encoding the NiV minigenome and N, L, and P WT and alanine scanning mutants. Induction levels were determined as described in the legend to Fig. 1. Western blots below the graph indicate the expression levels of the P protein constructs. (B) P WT and alanine scanning mutant plasmids were assayed for inhibition of IFN signaling as described in the legend to Fig. 2. IB, immunoblot.
- FIG. 5.
Single amino acid substitutions decrease NiV P's inhibition of IFN signaling and STAT1 binding. HA NiV P constructs harboring the indicated point mutations were assayed for polymerase cofactor activity, IFN signaling inhibition, and STAT1 binding as described in the legends to Fig. 1, 2, and 3, respectively. (A, D) 293T cells were cotransfected with the indicated P plasmids and pISG54-firefly luciferase. At 24 hpt, cells were either mock treated or treated with 1,000 U of IFN-β. At 16 h posttreatment, cells were lysed and analyzed for luciferase induction. Induction levels are shown as a ratio of firefly-to-Renilla luciferase levels. (B, E) Interaction of STAT1 with NiV P point mutants was determined in 293T cells transfected with the indicated HA-tagged P plasmids. Immunoprecipitations (IP) were performed with an anti-HA antibody, and Western blotting for STAT1 was performed. (C, F) BSR-T7 cells were cotransfected with NiV GFP-CAT minigenome, N, L, and P (WT and mutant) plasmids. At 24 hpt, cells were lysed and analyzed for CAT activity. Wedges represent 50, 100, and 200 ng of transfected P plasmid. IB, immunoblot; WCL, whole-cell lysate.
- FIG. 6.
Point mutant forms of NiV V and W do not interact with or inhibit STAT1. (A) Plasmids encoding the HA-tagged NiV V and W WT or mutant proteins were transfected in 293T cells and immunoprecipitated (IP) as described in the legend to Fig. 3. (B) IFN-β-induced STAT1 phosphorylation was assessed in 293T cells transfected with WT or G121E mutant versions of P, V, and W as described in the legend to Fig. 3C. IB, immunoblot; WCL, whole-cell lysate.
- FIG. 7.
Growth kinetics of WT and mutant NiVs. (A) Illustration of the recombinant NiV genome used to generate WT, Cko, and G121E mutant NiVs. Triangles indicate nucleotide changes designed to knock out the expression of the C ORF, and the star indicates the substitution creating the G121E mutation in the P, V, and W proteins. Growth kinetics of recombinant NiVs in 293T cells (B) and Vero E6 cells (C). Cells were infected at an MOI of 0.05, and titers in the culture supernatants were assessed at 1, 2, and 3 days postinfection. TCID50, 50% tissue culture infective doses.
- FIG. 8.
Impact of WT and mutant NiVs on the localization and phosphorylation of endogenous STAT1. Vero E6 cells were infected with the indicated viruses and treated with IFN-β at 12 h postinfection. The phospho-Y701 form of endogenous STAT1 (P-STAT1, green staining) was analyzed in infected cells paraformaldehyde fixed 40 min after IFN-β addition. NiV M protein expression is a marker for infection (red), and DAPI staining indicates the cell nucleus (blue). Arrowheads point to representative infected cells.
- FIG. 9.
WT and Cko viruses direct STAT1 to the nucleus, but the G121E mutant virus fails to control STAT1 localization. STAT1-GFP localization following NiV infection and subsequent IFN treatment. Vero E6 cells were transfected with STAT1-GFP plasmid, and at 10 hpt, cells were mock infected or infected with WT, Cko, or G121E mutant NiV. At 17 h postinfection, cells were treated with IFN-β and imaged at 40 min or 24 h posttreatment.
