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. 2008 Jan;82(1):529-37.
doi: 10.1128/JVI.02010-07. Epub 2007 Oct 17.

Accumulation of substrates of the anaphase-promoting complex (APC) during human cytomegalovirus infection is associated with the phosphorylation of Cdh1 and the dissociation and relocalization of APC subunits

Karen Tran  1 , Jeffrey A Mahr, Jiwon Choi, Jose G Teodoro, Michael R Green, Deborah H Spector
Affiliations

Accumulation of substrates of the anaphase-promoting complex (APC) during human cytomegalovirus infection is associated with the phosphorylation of Cdh1 and the dissociation and relocalization of APC subunits

Karen Tran et al. J Virol. 2008 Jan.

Abstract

Cell cycle dysregulation upon human cytomegalovirus (HCMV) infection of human fibroblasts is associated with the inactivation of the anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase, and accumulation of its substrates. Here, we have further elucidated the mechanism(s) by which HCMV-induced inactivation of the APC occurs. Our results show that Cdh1 accumulates in a phosphorylated form that may prevent its association with and activation of the APC. The accumulation of Cdh1, but not its phosphorylation, appears to be cyclin-dependent kinase dependent. The lack of an association of exogenously added Cdh1 with the APC from infected cells indicates that the core APC also may be impaired. This is further supported by an examination of the localization and composition of the APC. Coimmunoprecipitation studies show that both Cdh1 and the subunit APC1 become dissociated from the complex. In addition, immunofluorescence analysis demonstrates that as the infection progresses, several subunits redistribute to the cytoplasm, while APC1 remains nuclear. Dissociation of the core complex itself would account for not only the observed inactivity but also its inability to bind to Cdh1. Taken together, these results illustrate that HCMV has adopted multiple mechanisms to inactivate the APC, which underscores its importance for a productive infection.

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Figures

FIG. 1.
FIG. 1.
Cdh1 becomes phosphorylated during HCMV infection. HFFs infected with HCMV Towne at a multiplicity of infection of 5 (V) or incubated with conditioned medium (M) were harvested at 24 h p.i. Cell lysates were incubated with or without (−) λpp and were analyzed by Western blotting with antibodies to Rb, IE2, IE1, and Cdh1. Actin served as a control for protein loading. For Rb, IE2, and Cdh1, the phosphorylated forms are indicated with an asterisk.
FIG. 2.
FIG. 2.
Roscovitine treatment at early times after infection inhibits Cdh1 accumulation but not phosphorylation. (A) DMSO (−) or 20 μM roscovitine (+) was added at 0, 4, or 8 h p.i. to mock-infected (M) or HCMV-infected (V) cells. Cells were harvested at 24 h p.i. for Western blot analysis with antibodies to Rb, IE2, IE1, Cdh1, Cdc6, and geminin. Actin served as a loading control. For Rb and Cdh1, the phosphorylated forms are indicated with an asterisk. The Cdh1 blot for the 0- to 24-h-p.i. samples is slightly overexposed to show the presence of phosphorylated Cdh1 in the infected cells that were treated with roscovitine. (B) Mock-infected (M) or HCMV-infected (V) cells were treated with 20 μM roscovitine from 8 to 24 h p.i., harvested at 24 h p.i., incubated with or without (−) λpp, and analyzed by Western blotting as previously described.
FIG. 3.
FIG. 3.
Exogenous Cdh1 binding to APC3 from HCMV-infected cells is reduced as the infection progresses. Mock-infected (M) or HCMV-infected (V) HFFs harvested at 8 and 16 h p.i. were lysed and incubated with TNT-synthesized 35S-Cdh1 using APC-depleted reticulocyte lysate. Complexes were immunoprecipitated with antibody against APC3. Samples were assayed for APC3 by Western blotting (WB) and for coprecipitating 35S-Cdh1 by autoradiography. Short and longer exposures of the APC3 blot are shown. Empty vector pcDNA3 (p3) and IP using mouse IgG were used as negative controls. Pre, input lysate; IgG, IgG IP; IP, APC3 IP; Post, post-IP.
FIG. 4.
FIG. 4.
Levels of some APC core subunits increase during HCMV infection. Mock-infected (M) or HCMV-infected (V) HFFs were harvested at the times indicated and were processed by Western blot analysis with antibodies to APC3, APC7, APC8, APC10, APC11, and APC1. Actin was used as a loading control.
FIG. 5.
FIG. 5.
APC becomes destabilized during the course of HCMV infection. Mock-infected (M) or HCMV-infected (V) HFFs were harvested at the times shown and were immunoprecipitated for APC3. Western blots were used to identify coprecipitating APC subunits with antibodies to APC1, APC3, APC8, APC7, and Cdh1. The Cdh1 blot for the 8-h time point is slightly overexposed to show the presence of coprecipitating Cdh1 in the infected cells. GAPDH is shown as a negative control. Pre, input lysate; PC, IgG IP; IP, APC3 IP; PIP, post-IP.
FIG. 6.
FIG. 6.
Several APC subunits are relocalized to the cytoplasm of HCMV-infected cells. Mock- or HCMV-infected HFFs at 8, 12, and 16 h p.i. were fixed and assayed by immunostaining for APC1, APC3, and APC7. Mouse and rabbit IgG were used as negative controls on HCMV-infected cells from the 8-h-p.i. time point. Samples were analyzed by deconvolution microscopy using a 100× oil immersion lens, with pictures taken at 0.2-μm sections along the z axis. A midsectional plane of representative cells is shown. Images are separated to show the staining of each individual subunit; merged images also are shown with Hoechst staining in blue.
FIG. 7.
FIG. 7.
APC is inactivated through multiple mechanisms upon HCMV infection. (A) Schematic diagram of activated APC. Subunits are numbered accordingly. Unphosphorylated Cdh1 associates with and activates the complex, allowing the ubiquitination of the recruited substrate in concert with E1 and E2 ubiquitination enzymes. The relative location of the subunits is based on the model presented by Thornton et al. (38). (B) Model of APC upon HCMV infection. Cdh1 is phosphorylated (P) and no longer associates with the complex. APC1 becomes dissociated from the complex. It is unclear whether the subunits other than APC3, APC7, and APC8 (shaded) remain in complex together.
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