FIG. 3.

VSV P protein phosphorylation is altered in neuronal cell lysates, but not virions from IFN-β-treated cells. Neuroblastomas treated for 24 h with 400 U/mL IFN-β or vehicle alone were infected with VSV (MOI = 3), then either labeled with ³²P-orthophosphate (50 μCi/mL), or dual-labeled with ³²P-orthophosphate (50 μCi/mL) and ³⁵S-cysteine/methione (100 μCi/mL). At 6 hpi, cell lysates were harvested and immunoprecipitated with anti-VSV P antiserum. Samples were then resolved via 12% SDS-PAGE, stained via Coomassie Brilliant Blue, and exposed to film. (A) Example of a typical autoradiograph from anti-VSV P immunoprecipitated ³²P-labled cell lysates, along with Coomassie-stained IgG heavy chain as a loading control. Lane 1 represents viral proteins from untreated, uninfected cells; lanes 2–4 represent viral proteins from untreated, VSV-infected samples from independent experiments; lane 5 represents viral proteins from IFN-β-treated, uninfected cells; lanes 6–8 represent viral proteins from IFN-β-treated, VSV-infected samples from independent experiments. (B) Analysis of ³²P and ³⁵S metabolically incorporated into P protein from dual-labeled cell lysates. Using the autoradiograph as a guide, bands corresponding to the VSV P protein were excised, dissolved in 30% H₂O₂, and added to scintillation fluid (1:6) for counting. The normalized ratio of ³⁵S-associated counts (representing total protein) to ³²P-associated counts (representing phosphorylated amino acids; ³⁵S:³²P) from purified P protein increased in neuronal lysates derived from IFN-β-pretreated cells, representative of hypophosphorylation. Analogous experiments carried out in (C) NIH/3T3 and (D) L929 cells were unable to assess any potential changes in ³⁵S:³²P, due to a complete shutdown in protein synthesis resulting from an IFN-β-established antiviral state in these cells (denoted by asterisks). Error bars correspond to 95% CI and the p-values were calculated based on Satterthwaite's method for calculating t-values from independent samples of unequal variances (n = 12) (No T + V, no treatment + virus; IFN-β + V, IFN-β + virus).