FIG 2.

HCMV binding and entry are blocked in hESC. (A and B) hESC (panels I and II) and control human foreskin fibroblasts (panels III) were mock infected (panels I) or infected with HCMV strain TB40/E expressing pp150-fused GFP or strain TB40/E expressing pp65-fused GFP (panels II and III, respectively) at a multiplicity of infection of 1 PFU/cell. At 24 h postinfection, the cells were fixed, immunostained for HCMV pp65, and visualized by confocal microscopy (A) or directly visualized by fluorescent-phase microscopy (B). Large photos are at ×40 magnification (bar, 50 μm); small photos are at ×20 magnification (bar, 100 μm). A similar lack of pp65 internalization in hESC was observed at sequential time points between 2 and 48 h postinfection (viral incubation times equaled inspection times for these experiments; data not shown). (C and D) Quantitative viral DNA measurements, performed as described previously (27) in the indicated cell lysates, normalized by RNase P (RnP) gene DNA copies. (C) Viral entry assay. Cells were inoculated with HCMV strain TB40/E (multiplicity of infection of 0.5 PFU/cell) in the presence or absence of heparin, incubated at 37°C for 2 h, and treated with trypsin-EDTA to remove cell-bound noninternalized virus, and viral DNA quantity was analyzed by quantitative real-time PCR (27). (D) Viral binding assay. Cells were incubated with HCMV strain TB40/E (multiplicity of infection of 1 PFU/cell) at 4°C for the indicated times, in the presence or absence of heparin, collected by gentle scraping, and analyzed for viral DNA quantity as described above. Panel II shows the viral DNA accumulation kinetics over the 2 h of incubation. The minimal viral DNA accumulation in hESC, detected in panels C and D, is attributed to low-level viral infection which was limited to the small fraction of spontaneously differentiating cells (data not shown) that are commonly present in hESC cultures (22, 50). The asterisks indicate undetectable levels.