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. 2015 Jul;89(13):6928-39.
doi: 10.1128/JVI.00403-15. Epub 2015 Apr 22.

Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase-Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Downmodulation of the APC/C Subunits APC1, APC4, and APC5

Alex E Clark  1 , Deborah H Spector  2
Affiliations

Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase-Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Downmodulation of the APC/C Subunits APC1, APC4, and APC5

Alex E Clark et al. J Virol. 2015 Jul.

Abstract

Human cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C coactivator Cdh1 and by inducing the degradation of subunits APC4 and APC5, which along with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells, UL21a but not UL97 can disrupt APC/C function, leading to the accumulation of substrates. We find that UL21a is necessary and sufficient to induce the degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon the depletion of any one of these subunits or of subunit APC8. Finally, we show that at a low multiplicity of infection, either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by the expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using small interfering RNAs against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit downmodulation.

Importance: HCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in the control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform subcomplex of the APC/C, is an additional target of the degradation induced by HCMV protein UL21a. This study also shows for the first time that there is a unique cellular process in uninfected cells whereby depletion of APC1, APC4, APC5, or APC8 recapitulates the pattern of HCMV-mediated APC/C subunit degradation.

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Figures

FIG 1
FIG 1
Creation of UL21a and UL97-expressing fibroblasts. (A) EF1α promoter drives constitutive expression of a loxP-flanked selectable marker (selected by neomycin for UL21a and puromycin for UL97). Cre recombinase induces the expression of viral proteins via removal of the selection marker. (B) Cell-permeable Cre induces the expression of UL21a and UL97. Complementing cell lines plated on coverslips were treated with Cre and harvested 72 h later. Coverslips were stained with UL97 and UL21a antibodies and counterstained with Hoechst to visualize nuclei. Veh, vehicle.
FIG 2
FIG 2
UL21a but not UL97 expressed in uninfected cells promotes the accumulation of APC/C substrates. HF, HF-21a, HF-97, and HF-21a/97 cells were treated either with cell-permeable Cre to induce viral protein expression or with vehicle control for 12 h. Cells were then washed several times with PBS and maintained in HF medium. Cells were harvested 72 h after the beginning of Cre treatment and processed for Western blotting with antibodies to UL97, UL21a, geminin, Cdc6, Cdh1, APC1, APC4, APC5, APC6, and APC8. Tubulin served as a loading control. HF cells were infected at an MOI of 3 with WT HCMV (+) and harvested 24 hpi for comparison. Results for two different HF-21a/97 cell lines are shown; the cells whose blots are shown on the left had lower levels of expression of UL21a than those whose blots are shown on the right.
FIG 3
FIG 3
UL21a is necessary for APC1 degradation during infection. (A) Confirmation of anti-APC1 antibody specificity. (Left) Cells were mock infected (M) or infected with WT at high MOI and harvested 24 hpi for Western blotting with Ab to APC1. For molecular weight comparison, untreated HF cell lysate was immunoprecipitated with Ab to APC3 (IP) and run on the same gel. (Right) Cells were transfected with APC1 or nontargeting control (NT) siRNA. Four days posttransfection, cells were harvested and processed for Western blotting. (B) HF cells were confluence synchronized and mock infected or infected at high MOI with WT or Δ21a virus. Cells were harvested 24 hpi and processed for Western blotting with antibodies to UL21a, UL97, APC1, APC4, and APC5. Tubulin served as a loading control. (C) HF-21a cells were confluence synchronized and treated with cell-permeable Cre and/or 100 nM salinosporamide A (SalA) proteasome inhibitor as indicated. Cells were harvested 18 h posttreatment and processed for Western blotting with Ab to UL21a, APC1, APC5, and tubulin.
FIG 4
FIG 4
Knockdown of APC1, APC4, APC5, or APC8 causes the degradation of APC1, APC4, and APC5 in uninfected cells. (A) HF cells were transfected with APC1 (1), APC5 (5), or nontargeting control (NT) siRNA or left untransfected (−). Cells were harvested 96 h posttransfection and processed for Western blotting with antibodies to APC1, APC4, APC5, APC6, and APC8. (B) RNA was extracted from the samples used for the experiment whose results are shown in panel A, and real-time RT-PCR was performed to detect the levels of APC1 and APC5 transcripts. Transcript levels were normalized to the level of the tubulin transcript. Error bars indicate standard deviations of the results from three replicates of a single experiment. (C) HF cells were transfected with APC3 (3), APC4 (4), APC6 (6), APC8 (8), or nontargeting (NT) control siRNA or left untransfected (−). After 96 h, the cells were harvested and processed for Western blotting as described for panel A.
FIG 5
FIG 5
Creation of HCMV UL21a/UL97 double mutant. (A) Diagram of BAC recombinants. UL21a alone or UL21a and UL97 open reading frames in the wild-type (WT) Ad169 BAC HB5-IE-Cre/1.2-Flp (24) (referred to in all figures as WT) were replaced with ampicillin (UL21a) and tetracycline (UL97) resistance genes, respectively, and propagated on the HF-21a/97 complementing cells. (B) BamHI restriction digest of BAC recombinants and the molecular weight marker (MW) were resolved on an agarose gel. Arrows 1 and 2 indicate band changes predicted for mutation of UL97 and UL21a, respectively.
FIG 6
FIG 6
Δ21a/97 virus is defective for growth and deficient in late protein production. HF cells were confluence synchronized and mock infected (M) or infected with Δ21a/97, Δ21a, or WT virus at high MOI. (A) Supernatants were harvested 48, 72, 96, and 120 hpi and had their titers determined on HF-21a/97 cells. Shown are representative results of at least 2 independent experiments. Error bars indicate standard deviations. (B) Cells were harvested 24, 48, and 72 hpi and processed for Western blotting with antibodies to UL21a, UL97, IE proteins (IE2 86 and IE1 72), early protein (UL57), and late proteins (pp28 and IE2 40).
FIG 7
FIG 7
Cells infected with Δ21a/97 virus show a delay in the accumulation of APC/C substrates compared to their accumulation in cells infected with WT and Δ21a virus. HF cells were confluence synchronized and mock infected (M) or infected with Δ21a/97, Δ21a, or WT virus at an MOI of 4. Cells were harvested 24, 48, and 72 hpi and processed for Western blotting with antibodies to Cdh1, securin, geminin, and Cdc6.
FIG 8
FIG 8
The relative requirements for exogenous expression of UL97 and UL21a in complementing the growth of double mutant Δ21a/97 virus are MOI dependent. HF, HF-21a, HF-97, and HF-21a/97 cells were confluence synchronized and infected at an MOI of 3 (A, C) or an MOI of 0.1 (B, D). Supernatants were collected, their titers were determined on HF-21a/97 cells (A, B), and cells were harvested 96 hpi and processed for Western blotting with antibodies to UL21a, UL97, IE2 proteins (IE2 86, IE2 60, and IE2 40), and pp28 (C, D). Shown are representative results from 2 or 3 independent experiments. Error bars indicate standard deviations.
FIG 9
FIG 9
Knockdown of APC5 or APC8 results in increased growth of Δ21a/97 virus. (A) HF cells were transfected with APC5 (5), APC8 (8), or nontargeting control (NT) siRNA or left untreated (−). At 72 h after transfection, some cells were replated at lower density to release them into G1 phase (Released), while other cells were maintained at confluence (Confluent). The untreated cells were maintained in a cycling state (Cyc) by passaging them several times prior to harvest. At 96 h posttransfection, cells were harvested and processed for Western blotting with antibodies against APC5, APC8, cyclin A2, and tubulin. (B, C) HF cells were transfected with siRNAs as described for panel A. At 96 h after transfection, cells were mock infected (B). (C) Cells were mock infected or infected at an MOI of 0.5 with Δ21a/97 or WT virus. Cells were harvested 96 hpi and processed for Western blotting with antibodies to APC5, APC8, geminin, and Cdc6 (B) and antibodies to IE2 proteins (IE2 86, IE2 60, and IE2 40) and pp28 (C). The asterisk indicates another possible form of IE observed with the current lot of antibody. (D) Cell supernatants were harvested, and their titers were determined on HF-21a/97 cells. Shown are the results of two independent experiments.
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