Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97

Abstract

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

Keywords: cancer; cyclin-dependent kinase (CDK); cytomegalovirus; herpesvirus; oncogene; phosphorylation; retinoblastoma protein (pRb, RB); transcription.

FIGURE 1.

UL97 phosphorylates Rb on 11 CDK consensus sites during HCMV infection. A, serum-starved HFFs were mock-infected (M) or infected with wild-type (WT) or UL97-null (Δ97) HCMV. At the indicated hour postinfection (hpi), protein lysates were harvested and analyzed by Western blotting with the indicated antibodies. B, Saos-2 cells were transfected with expression plasmids for FLAG-tagged alleles of wild-type Rb or Rb in which CDK consensus phosphorylation residues were replaced with alanines (RbΔCDK) together with either an empty vector (EV) or one expressing HA-tagged WT UL97 or a kinase-deficient (KD) UL97. Lysates harvested 48 h after transfection were separated by conventional (normal) or phosphate affinity (Phos-tag) electrophoresis and analyzed by Western blotting with the indicated antibodies. pS, phosphoserine; pT, phosphothreonine.

FIGURE 2.

UL97 kinase activity is unaffected by mutation of the LXCXE motifs. A, Saos-2 cells were transfected with expression plasmids for Rb together with either an empty vector (EV) or HA-tagged alleles of wild-type UL97 or UL97 alleles with the following mutations: L1, C151G; L2, C428G; L3, C693G; Tri, C151G/C428G/C693G; HP, W368A; Qd, C151G/W368A/C428G/C693G; kinase-deficient (KD), K355Q. Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. B, U-2 OS cells were transfected with an expression plasmid for a lamin A-GFP fusion protein together with an expression plasmid for the indicated HA-tagged UL97 wild-type or mutant allele. UL97-expressing cells (as determined by indirect immunofluorescence with the HA antibody) were scored for having an intact or disrupted nuclear lamina after visualizing GFP fluorescence. The percentage of UL97-positive cells displaying a disrupted lamina is shown. Error bars represent the S.D. from biological triplicate. C, low multiplicity growth curves in HFFs were quantitated by relative light units (RLU) of secreted alkaline phosphatase (SEAP) activity. See “Experimental Procedures” for details. Error bars represent the S.E. of 11–12 replicate growth curves set up over three separate dates.

FIGURE 3.

The RXL motif of Rb is dispensable for Rb phosphorylation by UL97. A, Saos-2 cells were transfected with expression plasmids for HA-tagged alleles of wild-type Rb or Rb in which the C-terminal 99 amino acids containing the RXL motifs were deleted (RxL) together with either an empty vector (EV) or one expressing V5-tagged WT UL97. Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. B, Saos-2 cells were transfected with a luciferase reporter driven by the E2F1 promoter together with an expression plasmid for UL97 and either wild-type or RXL mutant Rb. Lysates harvested 48 h after transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb or UL97 (set at 100%). Error bars denote the S.D. **, p < 0.01.

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.

FIGURE 5.

Phosphorylation by UL97 relieves Rb-mediated repression of E2F-responsive promoters. A, Saos-2 cells were transfected with a luciferase reporter driven by the E2F1 promoter together with an empty vector (−) or an expression plasmid for Rb and the indicated allele of UL97. Lysates harvested 48 h after transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb or UL97 (set at 100%). Error bars denote the S.D. *, p < 0.05; **, p < 0.01; n.s., not significant. B, luciferase and Western blot analyses were performed as in A except an Rb allele in which CDK consensus phosphorylation residues were replaced with alanines (RbΔCDK;Δ) was also included. C, luciferase and Western blot analyses were performed as in A except the reporter contained an E2F1 promoter in which the E2F binding sites were mutated. D, luciferase and Western blot analyses were performed as in A except the reporter contained the Orc1 promoter. E, luciferase and Western blot analyses were performed as in A except the reporter contained the cyclin A promoter. F, luciferase and Western blot analyses were performed as in A except an Rb allele with an N757F CM was also included. KD, kinase-deficient.

FIGURE 6.

Underphosphorylation of Rb by UL97-L1m neither mediates sustained repression of E2F-responsive promoters by Rb nor affects disruption of Rb-E2F complexes. A, Saos-2 cells were transfected with a luciferase reporter driven by the E2F1 promoter together with an empty vector (−) or an expression plasmid for Rb alleles with specific phosphomimetic residue substitutions (3E, T373E/T821E/T826E; 3D, T373D/T821D/T826D) and the indicated allele of UL97. Lysates harvested 48 h after transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb or UL97 (set at 100%). Error bars denote the S.D. **, p < 0.01. B, luciferase and Western blot analyses were performed as in A except WT Rb or Rb alleles with different phosphomimetic (3Eb, T356E/T821E/T826E) or non-phosphorylatable (3A, T356A/T821A/T826A) residue substitutions were also included. C, Saos-2 cells were transfected with expression plasmids for wild-type FLAG-tagged Rb and the indicated allele of UL97. Lysates harvested 48 h after transfection were subjected to immunoprecipitation (IP) with the FLAG antibody. Input lysates and immunoprecipitates were analyzed by Western blotting with the indicated antibodies. h.c., heavy chain. D, immunoprecipitation experiment as in C except an Rb allele in which CDK consensus phosphorylation residues were replaced with alanines (Δ) was transfected. E, immunoprecipitation experiment as in C except the E2F1 antibody was used for immunoprecipitation. KD, kinase-deficient.

FIGURE 7.

Knockdown of E2F4 or p107 and p130 does not allow UL97-L1m to relieve Rb-mediated suppression of E2F-responsive transcription. A, Saos-2 cells were transfected with an siRNA targeting E2F4 or a control siRNA for 24 h. Cells were then transfected with a luciferase reporter driven by the E2F1 promoter together with an empty vector (−) or an expression plasmid for Rb and the indicated allele of UL97. Lysates harvested 48 h after plasmid transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb or UL97 (set at 100%). Error bars denote the S.D. *, p < 0.05; **, p < 0.01. B, luciferase and Western blot analyses were performed as in A except cells were transduced with lentiviruses expressing shRNAs targeting either GFP or p107 and p130. Luciferase activity is presented relative to the activity of the reporter without Rb or UL97 in each transduced cell (set at 100%). KD, kinase-deficient.

FIGURE 8.

The first UL97 LXCXE motif is dispensable for expression of the E2F-responsive genes E2F1, MCM7, and Cdt1 during HCMV infection likely because of complementation from the viral IE1 protein. A, 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. B, Western blot analysis conducted as in A except during either WT or UL97-null (Δ97) HCMV infection. C, E2F1 and MCM7 protein levels at 48 h postinfection from the experiment shown in A were quantified and normalized to GAPDH protein, and values are presented relative to the value in wild-type HCMV-infected cells (set at 1). Error bars denote the S.D. **, p < 0.01; n.s., not significant. D, serum-starved HFFs were infected with each virus at a multiplicity of infection of 2 in the presence of maribavir (MBV) or DMSO (D). E2F1 and Cdt1 mRNA levels at 48 h postinfection were analyzed by quantitative PCR and normalized to GAPDH mRNA level. Values are presented relative to the value in mock infection (M) (set at 1). Error bars denote S.D. *, p < 0.05; **, p < 0.01; n.s., not significant. E, Saos-2 cells were transfected with a luciferase reporter driven by the E2F1 promoter together with an empty vector (−) or an expression plasmid for Rb, the indicated allele of UL97, or HCMV IE1. Lysates harvested 48 h after transfection were analyzed for luciferase activity (top) and protein expression with the indicated antibodies (bottom). Luciferase activity was normalized to total protein concentration and is presented relative to the activity of the reporter without Rb, UL97, or IE1 (set at 100%). P-Rb, hyperphosphorylated Rb; non-P-Rb, non-phosphorylated Rb. F, Saos-2 cells were transfected as in E. Lysates harvested 48 h after transfection were analyzed by Western blotting with the indicated antibodies. G, model for Rb inactivation by UL97. I, Rb represses E2F-responsive transcription. II, UL97 induces E2F-responsive transcription both by Rb phosphorylation-dependent disruption of Rb-E2F complexes, which is an LXCXE-independent event, and by an unknown L1 LXCXE-dependent function. III, a UL97 mutant with a non-functional L1 motif (UL97-L1m) induces partial phosphorylation of Rb and disruption of Rb-E2F complexes but fails to activate Rb-repressed E2F-responsive transcription. IV, the inability of UL97-L1m to activate Rb-repressed E2F-responsive transcription is complemented by HCMV IE1 through an unknown mechanism. KD, kinase-deficient.