Virus preparation.

HCMV (strains Towne/E [passages 38 to 41] [98] and TB40-UL32-HCMV/E [99]) was cultured in human embryonic lung fibroblasts in Dulbecco's modified Eagle medium (DMEM) with 4% fetal bovine serum (FBS) (Mediatech, Inc., Manassas, VA) (16, 31, 35,–46, 51, 100,–102). The supernatants were collected, and virus was purified through a 0.5 M sucrose gradient and then resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Mediatech, Inc.) (16, 31, 35,–46, 51, 100,–102). Cells were infected at a multiplicity of infection (MOI) of 10 unless otherwise specified. Mock infection of cells was achieved using equivalent volumes of virus-free RPMI 1640. TB40-UL32-HCMV/E is a recombinant virus expressing enhanced green fluorescent protein (EGFP) fused to the C terminus of the capsid-associated tegument protein pUL32 (pp150) (43, 52, 99, 100). Because GFP is incorporated into a structural protein, viral particles can be tracked within the cell with no requirement for viral gene expression.