FIG 3.

Constitutive Akt activity causes defects in viral gene expression and viral DNA synthesis. (A) Fibroblasts were induced for expression of either myr-Akt or myr-Akt-K179M (kinase dead) for 24 h and then were infected with HCMV strain TB40/E at an MOI of 1, and total RNA samples were isolated from either myr-Akt or K179M settings at 2, 4, 6, 8, 10, 12, 16, 24, 48, and 72 hpi, including an on-column DNase I digestion step. RNA samples were reverse transcribed into cDNA and assayed by qPCR for the abundance of the indicated viral transcripts relative to levels of cellular GAPDH mRNA. (B) Total DNA was isolated at the indicated times postinfection (hpi) from cells infected exactly as described for panel A, and copies of viral DNA were enumerated using quantitative PCR for the HCMV UL69 gene normalized to copies of cellular 18S rDNA loci. (C) Infections were set up as described for panel A. Cell lysates collected at the indicated times postinfection were assayed by Western blotting for the expression of the indicated viral proteins, as well as for the expression of myr-Akt (HA tag) or GAPDH, as a loading control.