Figure 4. EC infection capacities correlate with pUL128 protein content of virus particles.

HFF, TIME cells and HUVEC were infected with vTB40-BAC4 or vBAC4-luc for the gradient purified virus as described in Materials and Methods to obtain equal numbers of initially infected cells (m.o.i. on HFF: 1). Supernatants were harvested, when cells showed about 80% CPE. (A) Supernatant-derived particles were concentrated by ultracentrifugation and pUL128 and gB protein levels detected by Western blot analysis of undiluted or 1∶4 diluted lysates. The pUL128 band intensities of undiluted samples are expressed as ratios of pUL128 band intensities to gB band intensities. This ratio was set to 100% for the HFF-derived particles and the ratios for HUVEC and TIME cell-derived particles expressed relative to the HFF value (middle panel). The virus preparations were additionally tested for their TIME/HFF infection ratios by staining infected HFF and TIME cells for ie1 protein expression 48 h after infection (lower panel). (B) Cellular expression levels of gB and pUL128 were monitored by Western blot analysis of total cell lysates of infected cells. (C) Analysis of gradient-purified virus derived from HFF and HUVEC infections. Cells were infected as described above. Virus was purified from the supernatants on glycerol/tartrate gradients as described in Materials and Methods and virus preparations analysed as described under (A).