FIGURE 4.

The first UL97 LXCXE motif and the Rb cleft mediate full phosphorylation of Rb by UL97. A, Saos-2 cells were transfected with an expression plasmid for Rb together with either an empty vector (EV) or one expressing HA-tagged WT UL97 or the indicated UL97 mutant allele. Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. For detection of phospho-Ser²⁴⁹/Thr²⁵², HFFs were serum-starved for 48 h and then stimulated with 15% serum. B, the graph represents the level of phosphorylated Rb normalized to the total Rb present from wild-type or L1m UL97-transfected cells shown in A. Values are presented relative to the value in wild-type UL97-transfected cells for each phosphorylation site (set at 1). Error bars denote the S.D. *, p < 0.05; **, p < 0.01; n.s., not significant. C, Saos-2 cells were transfected with expression plasmids for WT Rb or Rb with an N757F CM together with either an empty vector (EV) or one expressing V5-tagged wild-type UL97. Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. D, Lysates prepared as in C were treated (+) or not (−) with λ-protein phosphatase (l-PPase) and analyzed as in C. E, serum-starved HFFs were mock-infected (M) or infected with WT HCMV or the indicated UL97 mutant virus at a multiplicity of infection of 1. At the indicated hour postinfection (hpi), protein lysates were harvested and analyzed by Western blotting with the indicated antibodies. F, the graph represents the level of phosphorylated Rb normalized to the total Rb present from wild-type or L1m virus-infected cells at 48 h postinfection shown in E except at a multiplicity of infection of 2. Values are presented relative to the value in wild-type virus-infected cells for each phosphorylation site (set at 1). Error bars denote the S.D. *, p < 0.05; **, p < 0.01; n.s., not significant. pS, phosphoserine; pT, phosphothreonine; KD, kinase-deficient.