Article Figures & Data
Figures
- FIG. 1.
The attenuated mutant s51 (nsP1 538 Ile) grows to higher levels than the wild-type s55 (nsP1 538 Thr) virus in multistep in vitro growth curves. Neuro2A (A) or BHK-21 (B) cells were infected with s55 (solid squares) or s51 (solid circles) at an MOI of 0.01. Samples of supernatant were removed at the indicated time points and evaluated for virus yield by plaque assay. The data shown are from one of three representative experiments. Each point represents the average titer of three independent wells with the standard deviation.
- FIG. 2.
Accelerated nonstructural protein processing by the attenuated s51 virus in infected cells. BHK-21 cells were infected with the wild-type s55 (solid squares) virus (nsP1 538 Thr) or the attenuated s51 (solid circles) virus (nsP1 538 Ile) at an MOI of 5.0 for 2 h. The infected cells were then pulsed with [35S]methionine for 15 min followed by a chase with an excess of cold methionine for 10, 20, or 30 min, at which points, cell lysates were generated. (A) NsP1 was immunoprecipitated from the cell lysates and analyzed by SDS-PAGE on 10% polyacrylamide gels. Normal rabbit serum served as a control for nonspecific antibody interactions. (B) Plot of the relative levels of nsP1 from s55- or s51-infected cells from panel A. Shown are the results of one of four representative experiments.
- FIG. 3.
The attenuating Ile at nsP1 538 accelerates nonstructural protein processing in vitro. (A) Full-length RNA transcripts for s55 (solid squares) or s51 (solid circles) were in vitro translated in the presence of [35S]methionine by using rabbit reticulocyte lysates. Translation reactions were incubated for 40 min at 30°C. At this point, an excess of unlabeled methionine and cycloheximide was added to stop translation, and reactions were shifted to 37°C. Samples were removed for analysis by SDS-PAGE at 0, 20, 40, 60, and 80 min after stopping translation and increasing the temperature. (B) Levels of the P123 and P12 cleavage intermediates as well as mature nsP1 and nsP2 for the s55 or s51 translation reactions were analyzed over time on a phosphorimager. The data represent the total number of pixels for the relevant band at each time point. The data shown are from the gels pictured in panel A. (C) Full-length RNA transcripts for the attenuated TR339 (nsP1 538 Ile) and neurovirulent 39ns1 (nsP1 538 Thr) were translated as described for Fig. 2A. Shown are the results of one of three representative experiments.
- FIG. 4.
The attenuating Ile at nsP1 538 increases expression from the S.A.AR86 26S promoter. BHK-21 cells were infected with S.A.AR86-derived replicon particles with the wild-type Thr (REP89-GFP) or the mutant Ile (REP91-GFP) at nsP1 538 at an MOI of 0.1. These replicons express GFP from the viral 26S promoter, which was used as a surrogate marker of 26S expression. At 4 h postinfection, cells were harvested and analyzed for GFP expression (fluorescence [Fl]) by flow cytometry. Shown are representative histograms from one of three experiments with BHK-21 cells.
- FIG. 5.
The attenuating Ile leads to faster induction of 26S RNA synthesis, but does not affect minus- or plus-strand synthesis. BHK-21 cells were infected with the wild-type s55 or mutant s51 viruses at an MOI of 5.0. Total cellular RNA was harvested at 3 or 6 h postinfection and analyzed by RNase protection assays using probes protecting fragments from the viral plus and 26S RNAs, the viral minus-strand RNA, or mouse β-actin. Representative RNase protection assay showing the 3- and 6-h time points for s55- or s51-infected cells. Each lane represents RNA from an independent sample. Five micrograms of total cellular RNA was used for each sample at the 3-h time point, and 1 μg per sample was used for the 6-h time point. Minus-strand and β-actin bands are shown at different intensities due to the excess of β-actin signal compared to minus-strand levels.
Tables
- TABLE 1.
Ratios of s51 versus s55 RNA in infected cells
Cell type Time post- infection (h) s51/s55 RNA ratioa Minus strand Plus strand 26S RNA BHK-21 3 0.8, 0.8 1.1, 0.6, 1.0 4.2, 3.1, 4.2 4 1.3 1.2 1.4 5 0.8 1.0 1.0 6 0.9 1.2, 0.9 1.0, 1.0 Primary neurons 3 0.8 2.7 6 0.6 1.0 ↵ a Each data point represents the ratio of s51 to s55 RNA for a single experiment.
