Inactivation and disassembly of the anaphase-promoting complex during human cytomegalovirus infection is associated with degradation of the APC5 and APC4 subunits and does not require UL97-mediated phosphorylation of Cdh1

Abstract

Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G(1)/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved.

FIG. 1.

APC substrates accumulate during ΔUL97 virus infection. HFFs were mock-infected (M) or virus-infected with ΔUL97 virus (Δ) or AD169 (V) at an MOI of 3. (A) Cells were harvested over an infection time course and analyzed by Western blot assay for the expression of HCMV genes and cell cycle regulators. Actin is shown as a loading control. (B) Total RNA isolated from cells harvested over an infection time course was analyzed for geminin and G6PD expression by qRT-PCR. Geminin values were normalized to the expression of G6PD as a control for input RNA. Values are expressed as the fold induction over the level at 0 h p.i. (C) Cell samples were harvested at 16 h p.i., treated with or without (−) lambda protein phosphatase (λpp), and analyzed by Western blot assay. Equivalent cell numbers were loaded for each sample, except for lane 7 (†), which contained one-fifth the amount. Results for short and long exposures of the Cdh1 blot are shown. IE2 86 was a positive control for the phosphatase assay, while actin served as a negative and loading control. Lane numbers are shown below.

FIG. 2.

UL97 phosphorylates Cdh1 at multiple sites in vitro. (A) In vitro kinase assays using purified Cdh1 and GST-UL97 were performed with [γ-³²P]ATP in the presence or absence of maribavir (MBV). ³²P incorporation was imaged on a phosphor screen, and the Coomassie-stained gel of the samples is shown below as a loading control. (B) The phosphopeptides identified by MS are listed with the phosphorylated amino acid shown by boldface with a lowercase p, and the corresponding position numbers are indicated. Cdk consensus sites and those conforming to the UL97 preferential sequence (P+5) are indicated.

FIG. 3.

The APC dissociates with similar kinetics during ΔUL97 virus infection and wild-type virus infection. HFFs infected with ΔUL97 virus (Δ) or AD169 (V) at an MOI of 3 or mock-infected (M) were harvested at the times indicated. The APC was immunoprecipitated using an anti-APC3 antibody, and coimmunoprecipitated proteins were analyzed by Western blot assay with antibodies to APC3, APC7, APC8, APC1, and Cdh1. GAPDH is shown as a negative and loading control. Lane numbers are shown below. Pre, input lysate; IP, APC3 IP; PIP, post-IP.

FIG. 4.

Decreased APC4 and APC5 protein expression is observed during HCMV infection. (A) Levels of APC4, APC5, APC6, and APC3 protein expression were assessed by Western blot assay through an HCMV Towne (V) infection time course. GAPDH is shown as a loading control. (B) APC subunit expression was further examined in cells infected with ΔUL97 virus (Δ) and AD169 (V) by Western blot. Actin is shown as a loading control. (C) Total RNA was isolated from mock- or HCMV Towne-infected cells and analyzed for APC4, APC5, and G6PD expression by qRT-PCR. APC4 and APC5 values were normalized to the expression of G6PD as a control for input RNA. Values are expressed as the fold induction over the level at 0 h p.i.

FIG. 5.

APC4 and APC5 are degraded by the ubiquitin-proteasome pathway during HCMV infection. (A) HFFs infected with HCMV Towne (V) (MOI of 2) or mock-infected (M) were treated with proteasome inhibitor MG132 (2.5 μM, reversible) or Sal A (100 nM, irreversible) at 6 h p.i. Cells were harvested at 12 h p.i. or placed in fresh medium without drug and harvested at 24 h p.i. Samples were processed by Western blotting for protein expression. Lane numbers are shown below. (B) Mock-infected (M) or Towne-infected (V) cells were treated with Sal A (100 nM) or the E1 inhibitor PYR-41 (10 μM) at 6 h p.i. and harvested at 18 h p.i., and protein expression analyzed by Western blot assay.

FIG. 6.

APC dissociation is prevented with proteasome inhibitors. Mock-infected (M) and Towne-infected (V) HFFs (MOI of 2) were treated with or without Sal A (100 nM) from 6 to 14 h p.i. and harvested at 14 h p.i. APC3 co-IP assays were performed as previously described with coimmunoprecipitated proteins analyzed by Western blot assay. GAPDH is shown as a negative and loading control. Lane numbers are shown below. Pre, input lysate; IgG, IgG IP; PIgG, post-IgG; IP, APC3 IP; PIP, post-IP.

FIG. 7.

Viral tegument and IE protein expression alone are insufficient to cause the loss of APC4 and APC5 protein expression. HFFs were treated with mock-infected (M), virus-infected (V), or UV-inactivated (UVi) tissue culture supernatants and harvested at 2, 4, 6, and 12 h p.i. To assess the requirement for IE expression, HFFs infected with HCMV Towne (MOI of 2) were treated with 100 μg/ml CHX from 2 to 12 h p.i. (+2), 4 to 12 h p.i. (+4), or 6 to 12 h p.i. (+6) to inhibit synthesis of early proteins. Samples were analyzed by Western blot assay. Short and long exposures of the UL44 blot are shown, and lane numbers are indicated below.

FIG. 8.

Viral early gene expression is required to mediate the degradation of APC4 and APC5. Mock-infected (M) or Towne-infected (V) HFFs (MOI of 2) were treated with CHX (100 μg/ml) from 0 to 6 h p.i. and harvested at 6 h p.i. or washed and released into fresh medium or ActD (20 μM) and harvested at 18 and 24 h p.i. ActD (20 μM) was added at 6 h p.i., and cells were harvested at 18 and 24 h p.i. Lysates were analyzed by Western blot assay for APC4, APC5, IE, and UL44 expression. Actin is shown as a loading control. Lane numbers are shown below.

FIG. 9.

Inactivation of the APC during HCMV infection. (A) Schematic diagram of the APC^Cdh1. The essential mammalian APC core subunits are shown and numbered accordingly. (B) Model illustrating the mechanisms by which the APC is disabled during HCMV infection. (I) APC5 and APC4 are targeted for degradation by the proteasome and the APC dissociates, with the TPR subunits and APC10 localizing to the cytosol while APC1 remains nuclear. (II) Cdh1 is phosphorylated, mediated by UL97, and no longer associates with the complex.