FIG. 8.

The dependence of HCMV infection on state of cellular differentiation is due to a restriction at the level of viral entry. Infection studies using a recombinant HCMV (HV5.111) that constitutively expresses GFP were conducted to determine whether the restriction of HCMV infection in Caco-2 cells at late times of differentiation was at the level of virus entry; HV5.111 places GFP under the control of the constitutively active promoter of EF1α, an essential component of the protein translational machinery. HV5.111 was added at an MOI of 25 to either the apical (AP) or the basolateral (BL) media of Caco-2 cell monolayers at increasing states of cellular differentiation as measured by TER. At day 4 p.i. (time of maximal GFP expression), monolayers were fixed, and the level of infection was determined by visualization of GFP-positive cells with a Nikon Optiphot fluorescence microscope.