FIG. 4.

Phosphorylation of A17 is abrogated during nonpermissive infections with ts mutants defective in the F10 kinase. (A) Incorporation of ³²PP_i into A17 is not observed during nonpermissive infections with ts28 (tsF10). Confluent BSC40 cells were infected (MOI of 2) with wt virus or ts28 (tsF10) and maintained at the permissive (31.5°C) or nonpermissive (39.5°C) temperature. Cultures were metabolically labeled with ³²PP_i from 3 to 17 hpi, harvested, and subjected to immunoprecipitation with anti-A17 (lanes 1, 4, 7, and 10), anti-pTyr (lanes 2, 5, 8, and 11), or anti-I3 (lanes 3, 6, 9, and 12) serum. Incorporation of ³²PP_i into I3 (filled oval) was equivalent under all conditions, whereas incorporation of ³²PP_i into A17 (filled arrow) was lost in cells infected with ts28 at 39.5°C (compare lane 4 with lanes 1, 7, and 10). Since the H1 phosphatase was active during all of these infections, recovery of ³²P-labeled A17 with anti-pTyr serum (open arrow) was minimal. (B) A17 does not contain immunoreactive pTyr in cells infected with ts28 under nonpermissive conditions. Cells were left uninfected or infected with wt virus or ts28 (tsF10) under permissive and nonpermissive conditions (MOI of 10). At 12 hpi, cells were harvested and subjected to immunoblot analysis with anti-pTyr serum. All of the immunoreactive pTyr in the A17 protein (open arrow) was lost during nonpermissive infections with ts28 (compare lane 6 with lanes 3 to 5).