Fig. 5. cAMP/IBMX initiates HCMV replication.

T98G-LrV cells were treated with DMSO or cAMP/IBMX for about 30 days. Cells and supernatants were collected for following assays. (A) Viral replication centers. The cells were plated onto coverslips and examined the expression of GFP and UL44 by IFA. Scale bar: 10 µm. (B) Viral genome copy number. Total DNA was extracted from the collected cells, and viral genome copy number per cell was determined by qPCR. *** p < 0.001. (C) Transcription levels of the representative lytic viral genes. RNAs were extracted from the collected cells followed by RT-PCR, and relative transcription levels of the indicated viral genes were assessed by qPCR with GAPDH as the reference gene. (D) Viral proteins expression. The collected cells were lysed and subjected to western blotting for examination of the indicated viral proteins. Actin serves as the loading control. (E) Effect of PAA on IE1 transcription. T98G-LrV cells were pretreated with PAA for 12 days, then cultured for additional 8 days in the presence of DMSO or cAMP/IBMX. Total RNA was extracted, and IE1 transcription level was quantified by qPCR. The relative level of IE1 was calibrated to that in the DMSO treated cells in the presence of PAA, with GAPDH as the reference gene, and the uninfected T98G cells as the mock.