FIG. 5.

(A) F10 proteins encoded by ts15 and ts54 are thermolabile in vivo. Cells were infected with wt virus or the tsF10 mutant viruses at an MOI of 5 and maintained at either 31.5 or 40°C for 12 h. Extracts were subjected to immunoblot analysis with the anti-F10 antiserum; the relative amount of F10 protein seen in each lane was determined by spot densitometry. For each virus, the ratio of the amount of F10 accumulated at 40°C to that accumulated at 31.5°C is shown as a percentage below the relevant lanes of the immunoblot. (B) Recombinant His-ts28 F10 lacks detectable kinase activity in vitro. Purified N′ His-tagged versions of wt F10, ts28 F10 (Val₁₆₇→Ile), and ts15 F10 (Pro₁₃₁→Ser) were assayed in vitro for protein kinase activity, using the generic substrate MBP. For each enzyme preparation, three assays containing increasing amounts of enzyme (∼35 to 140 ng) were performed. Reactions were fractionated in an SDS-15% PAGE gel and analyzed by autoradiography (top); phosphorylated MBP is indicated by the arrow. The specific activities of the wt F10 and ts15 F10 preparations were comparable, whereas the ts28 F10 preparation had no detectable enzymatic activity (<2% of the wt activity). An immunoblot developed with a His probe (bottom) confirmed that similar amounts of the three preparations were used in the assays. The migration of protein standards is shown at the left, with molecular masses indicated in kilodaltons.