FIG. 7.

Alteration of the late phosphorylation pattern of T585A and S588A mutant forms of NS1. A9 cultures arrested in G₀/G₁ were infected with indicated viruses, released into the cell cycle for 48 h and metabolically ³²P labeled. NS1 proteins were isolated, purified, and processed for the analysis of tryptic phosphopeptides by two-dimensional electrophoresis and/or chromatography. Compared to wild-type NS1 (A), the NS1-585A (B) and NS1-588A (C) mutants lacked specific phosphorylation event(s), as evidenced by the disappearance of distinct phosphopeptide(s) (arrowheads). (D) Schematic representation of the “late” NS1 tryptic phosphopeptide pattern, in which the spots absent from the NS1-585A and NS1-588A maps are indicated with filled black and open circles, respectively, and the previously assigned PKCλ phosphorylation targets T435 and S473 are positioned.