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. 2012 Sep;86(17):9369–9383. doi: 10.1128/JVI.07181-11

Fig 4.

Fig 4

MCMV is insensitive to cyclin A2 overexpression and induces cyclin A2-associated kinase activity after infection of quiescent cells. (A) Cyclin A2ΔD and cyclin A2ΔD(R211A)-expressing U373 derivatives (see the legend to Fig. 1), as well as control cells, were infected with MCMV. Five hours postinfection, the cells were harvested and analyzed for IE1 protein expression and DNA content by flow cytometry. (B, C, and D) Mouse 3T3 fibroblasts were lentivirus transduced with expression vectors for human and mouse cyclin A2. (B) Flow cytometric analysis of EGFP marker expression was performed to document homogeneously transduced cell pools. (C) The expression of exogenous cyclin A2 protein was checked by HA immunoblot analysis. A nonspecific band is indicated by an asterisk. (D) Cyclin A2-overexpressing and control 3T3 fibroblasts were infected with MCMV and analyzed at 5 h postinfection for IE1 protein expression and cellular DNA content by flow cytometry. (E) Fibroblasts of human (HEL) and mouse (3T3) origin were made quiescent by 48 h of serum starvation. Then (at 0 h), the cells were either restimulated by 10% fetal calf serum containing medium (+ serum), CMV infected in the presence of medium without serum (+ HCMV/MCMV), or both restimulated and infected by adding virus and serum containing fresh medium simultaneously (+ HCMV/MCMV, + serum). Cells were harvested at regular intervals and prepared for analysis of their endogenous cyclin A2-, B1-, and E1-associated kinase activities. Shorter intervals were chosen for the murine system, because both the 3T3 cell division and the MCMV replication cycle proceeded faster than for their human counterparts. Cyclin-CDK complexes were isolated from cell extracts by immunoprecipitation (IP) using antibodies that are proven to specifically bind the indicated cyclins without destroying the associated kinase activity. To measure this kinase activity, in vitro kinase assays were performed using histone H1 as a substrate. Shown are autoradiographic images of histone H1 phosphorylation. The results are representative of more than three independent experiments.