Fig 2: Interaction with pUL21a leads to Cyclin A2 protein degradation.HEK293 cells were transfected with the indicated pUL21a-wt and mutant expression plasmids (pUL21a-PRAA: APCmut) or an empty vector control. Where indicated, MG132 was added to the cells at 24 h post transfection. Cells were harvested at 48 h post transfection and processed for the following assays. (A) Cyclin A2-CDK complexes were immunoprecipitated (IP) from extracts of transfected, MG132-treated cells and analyzed by immunoblotting for the presence of Cyclin A2, CDK2 and HA-pUL21a. (B) Whole cell lysates of transfected cells were analyzed by immunoblotting for expression of the indicated proteins. (C) Total RNA preparations of transfected cells were analyzed by quantitative RT-PCR for Cyclin A2 and APC5 mRNA expression. Error bars indicate the standard deviations of triplicate samples.

Fig 3: Working model of virus-mediated APC regulation during HCMV infection.HCMV uses two mechanisms to regulate the APC during infection. The pUL97 viral kinase inhibits the APC co-activator Cdh1 by phosphorylation while pUL21a targets APC4 and APC5 for proteasome-dependent degradation to dissociate the complex.

Fig 4: pUL21a reduces APC4 and APC5 protein levels and inhibits APC activity in an inducible cell line.(A) pUL21a expression is sufficient to reduce APC4 and 5 protein accumulation. Hela cells constitutively expressing GFP-tagged TetR were transduced with pLKO-derived lentivirus that expressed pUL21aPH-AA, pUL21astop, or pUL21aPR-AA under a Tet operator (TetO)-regulated CMV promoter. Transduced cells were enriched by antibiotic selection and pUL21a expression was induced by tetracycline treatment for 72 hours. Cell lysates were collected and analyzed by immunoblotting. (B) pUL21a expression is sufficient to induce an M-phase cell cycle arrest. Cells carrying pUL21aPH-AA (+/- tetracycline) were treated with nocodazole (100 ng/µl) for 16 hours to synchronize cells in G2/M phase. The cells were then released from nocodazole, collected at indicated time points, and analyzed for DNA content by flow cytometry. (C) pUL21a expression is sufficient to regulate APC activity. Cells from panel B were also analyzed for APC subunit and substrate levels by immunoblotting.