FIG. 5.

Transient rescue of vindA14 performed with Ser-to-Ala mutants of A14. (A) Determination of complementation competence by titration of viral yield from infections and transfections. Infection-transfection assays were performed as described above. Cells infected with vindA14 in the absence of inducer were transfected with empty vector, plasmid encoding wt A14, or plasmids encoding the various Ser-to-Ala mutants in order to assess their ability to substitute for endogenous A14. All experiments were performed in triplicate, and the titers of viral yield were averaged. The horizontal grey line illustrates the titer of virus obtained upon transfection of empty vector. (B) Immunoblot analysis of A14 expression. Aliquots of the extracts described above were resolved by nonreducing SDS-17% PAGE and were subjected to immunoblot analysis to monitor A14 expression from the endogenous and transfected alleles. The positions of the 18,000- and 29,000-M_r markers are shown at the left. (C) ³²PP_i labeling of A14 Ser-to-Ala mutants. Infections and transfections were performed as described earlier. Cells were metabolically labeled with ³²PP_i from 6 to 24 hpi; cell lysates were subjected to immunoprecipitation analysis with anti-A14 serum in order to determine the phosphorylation status of the various A14 proteins. In the case of wt and vA14(S85A) virus infections (rightmost lanes 1 and 2), cells were labeled with [³⁵S]methionine from 5 to 6 hpi and with ³²PP_i from 6 to 9 hpi prior to being harvested at 9 hpi and subjected to immunoprecipitation with anti-A14 serum as described above. In all cases, immunoprecipitates were resolved by SDS-PAGE and were visualized by autoradiography.