FIG. 3.

HCMV accumulates less IE2-86 protein and fewer IE transcripts at late times and synthesizes viral DNA at reduced levels in the absence of UL21a. (A) Analysis of viral protein accumulation. MRC-5 cells were infected with ADwt or ADsubUL21a at an input genome number equivalent to 0.25 (left panel) or 3 (right panel) TCID₅₀ units of wild-type virus/cell. Cells were collected at the indicated times, and total cell lysates were analyzed by immunoblotting with the indicated antibodies. (B) Analysis of viral transcript accumulation. MRC-5 cells were infected with ADwt or ADsubUL21a at an input genome number equivalent to 0.25 (left panel) or 3 (right panel) TCID₅₀ units of wild-type virus/cell. Total RNA was collected at the indicated times, and amounts of selected viral IE transcripts were measured by reverse transcription-coupled quantitative PCR (RT-qPCR) and normalized to that of GAPDH. Also shown are the genomic structure of the MIE locus and the locations of RT-PCR primers for analyzing IE1, IE2, and total IE transcripts. The normalized amount of viral transcript at 24 hpi during wild-type virus infection was set to 1. (C) Analysis of viral DNA synthesis. MRC-5 cells were infected with ADwt or ADsubUL21a at either an input genome number equivalent to 0.25 TCID₅₀ unit of wild-type virus/cell or an MOI of 0.05. Total intracellular DNA was collected, viral DNA was measured by qPCR, and the amount was normalized to cellular genome copies with primers to β-actin. The normalized amount of viral DNA at 2 hpi during wild-type virus infection was set to 1. Shown are representative results from at least two independent experiments. Error bars indicate standard deviations.