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. 2014 Nov 5;89(2):1070–1082. doi: 10.1128/JVI.01740-14

FIG 4.

FIG 4

Overexpression of Cdc25a enhances HCMV replication in U-251MG cells. (A) Potential base pairing is indicated by vertical lines between the seed sequence of miR-21 (bold) and its target sequences within the wild-type (WT) 3′UTR of Cdc25a. Mutation (MT) of the miR-21 target sequence is predicted to eliminate miR-21-mediated repression. (B) HEK293T cells were cotransfected with plasmids expressing miR-21 or control miRNA miR-UL22A, along with reporter plasmids encoding luciferase, followed either by the Cdc25a 3′UTR (Cdc25a) or a control 3′UTR (CCNE2). The luciferase activities were measured at 48 h posttransfection and are expressed as percent differences relative to cells cotransfected with the CCNE2 and miR-UL22A-expressing control plasmids. (C) For the left panel, HEK293T cells were cotransfected with a plasmid encoding Cdc25a, followed by its native 3′UTR with either a miR-UL22A-expressing control plasmid (4 μg) or a miR-21-expressing plasmid (2 or 4 μg). For the right panel, HEK293T cells were cotransfected with miR-21-expressing plasmid (4 μg) with either pCDH-Cdc25a+WT-3′UTR-GFP (2 μg) or pCDH-Cdc25a+MT-3′UTR-GFP (2 μg). Cdc25a protein levels were determined by Western blotting. Protein levels relative to miR-UL22A-transfected control cells were determined by densitometry and are indicated below each blot. Actin serves as a loading control. (D) U-251MG cells were transduced with lentiviruses expressing Cdc25a without its native 3′UTR (Cdc25a) or empty vector control (Ctrl, pCDH-GFP) and Cdc25a was detected by Western blotting. Protein levels relative to Ctrl-transduced cells were determined by densitometry and are indicated below each blot. Actin serves as a loading control. (E) Lentivirus-transduced U-251MG cells (Ctrl and Cdc25a) were infected with HCMV at an MOI of 1, and viral and cellular proteins were detected by Western blotting at the indicated times postinfection. Protein levels relative to Ctrl were determined by densitometry and are indicated below each blot. Actin serves as a loading control. (F) Titers of infectious virus in the culture supernatants at 72 hpi were determined by plaque assay. The fold differences are indicated. Luciferase and virus titer results are means ± 1 SD of data from three independent experiments, each conducted in triplicate. *, P < 0.05; **, P < 0.01.