Article Figures & Data
Figures
- Fig. 1.
(A) The SeV P gene expresses four C proteins. The unedited P gene mRNA is shown at the top and its three ORFs (P, C, and V) are shown as horizontal boxes drawn roughly to scale (above). A blow-up of the 5′ end of the mRNA is shown in the middle, and the five ribosomal start sites in this region are indicated. The numbers refer to positions from the 5′ end of the mRNA and to the first base of the start codon. An immunoblot of the C proteins expressed in BHK cells infected with SeVwt and SeV unable to express C′ or C due to mutation of ACG81 and AUG114 (27) is shown in the middle. The properties of the individual C proteins are summarized at the bottom. (B) Various pTM1-C protein expression plasmids. pTM1 contains a T7 RNAP promoter and generates mRNAs beginning with the EMCV IRES up to the CCAUGG start codon and NcoI restriction site (thick line at left with Y-shaped structure), which permits cap-independent translation. The various C genes cloned into pTM1, as described in the text, are shown schematically. The start codons in lowercase letters are silent (6).
- Fig. 2.
Effect of wild-type and mutant C gene expression on IFN-α signaling to pISRE-luc. Parallel cultures of BF cells were transfected with 1.5 μg of pT7 (which expresses cytoplasmic T7 RNAP from a CMV promoter), 1 μg of pISRE-luc, and 1.5 μg of one of the various pTM1-C as indicated. When transfections included two pTM1-C plasmids, all other reagents were doubled. At 48 h posttransfection, the cultures were treated or not treated with 1,000 U of IFN-α. The cultures were then metabolically labeled with Tran35S-label at 50 h posttransfection and harvested 2 h later, and the level of luciferase activity in the cell extracts was determined. The averages and ranges of activities from duplicate transfections of each pTM1 constructs and their mixtures are shown. Equal amounts of the various transfected cell extracts were also immunoprecipitated with an excess of anti-C antisera and then separated by SDS-PAGE. An autoradiogram of the gel is shown below. A time line of the experiment is shown above.
- Fig. 3.
Effects of the various C proteins on IFN-α signaling to pISRE-luc and IFN-γ signaling to pGAS-luc. Parallel cultures of BF cells were transfected with 1 μg of pT7 and 1 μg of pISRE-f.luc (A) or 1 μg of pGAS-f.luc (B) and 0.2 μg of pTK-r.luc to control for transfection efficiencies and 1 μg of one of the various pTM1-C as indicated (see also Fig. 1B). At 48 h posttransfection, the cultures were treated (or not) with 1,000 U of IFN-α. The cultures were then metabolically labeled with Tran35S-label at 58 h posttransfection and harvested 2 h later, and the levels of the firefly (f.luc) and renilla (r.luc) luciferase activities in the cell extracts were determined. The averages and ranges of the ratios of firefly to renila activities from duplicate transfections of each pTM1 construct are shown. Equal amounts of the various transfected cell extracts were also immunoprecipitated with an excess of anti-C antisera and separated by SDS-PAGE. (C) An autoradiogram of the gel is also shown. Note that the strain M and H AUG114-initiated C proteins migrate at slightly different rates, as highlighted by a short line which connects the MVC and H lanes. A time line of the experiment is shown above panel A.
- Fig. 4.
Effect of SeV infection and expression of the various C proteins on pISRE-luc activity. Parallel cultures of BF cells were transfected as described in Fig. 3. At 24 h posttransfection, the cultures were either not infected or infected with SeVM or SeVMVC, and treated (or not treated) with 1,000 U of IFN-α at 48 h posttransfection as indicated. The cultures were harvested at 60 h posttransfection, and the levels of the firefly (f.luc) and renilla (r.luc) luciferase activities in the cell extracts were determined. The averages and ranges of the ratios of activities from duplicate transfections of each pTM1 construct are shown in panel A. The experiment in panel B was carried out similarly to that in panel A, except that only SeVMVC infection was used, as indicated. A time line of both experiments is shown above.
- Fig. 5.
Effect of SeV infection on STAT1 levels and induction of an antiviral state. Parallel cultures of NIH 3T3 mouse embryo fibroblasts were infected with either SeVM, SeVMVC, SeVZ-CM (rM), or SeVZ-CMVC (rMVC) or were not infected with SeV and then concomitantly treated (or not) with 1,000 U of IFN-α as indicated. Most cultures were also superinfected with VSV at 50 h post-SeV infection, as indicated. The cultures were harvested at 55 h postinfection, and the relative levels of the cellular STAT1 and VSV P proteins were determined by immunoblotting. An anti-actin antibody was included to control for the amount of cellular material loaded on each lane. Equal samples of the various cell extracts were used to determine the relative levels of the SeV N and P proteins present by immunoblotting.
