The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes.

<p>(A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO₂. Monocytes were then mock- or HCMV (BAD<i>wt</i> or BAD<i>r</i>UL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003463#s2" target="_blank">Results</a> are plotted as a mean ±SEM. Student's T-tests were performed and p<0.05 (indicated by asterisks) was used for the measurement of statistical significance between samples. (C and D) Monocytes were mock- or HCMV (BAD<i>wt</i> or BAD<i>r</i>UL131)-infected [M.O.I. of 5 (C) or 1 (D)] and incubated at 37°C in 5% CO₂ for 5 days. (C) Cells were fixed, cytospun, underwent fluorescence <i>in situ</i> hybridization and were analyzed using a confocal microscopy (vDNA – yellow/green & DAPI nuclear stain – pink) or (D) were harvested and semiquantitative PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Note: in the (C) panel, inset pictures are an enlarged version of the representative cell shown in the picture. (E) Monocytes were HCMV (BAD<i>wt</i> or BAD<i>r</i>UL131)-infected (M.O.I. of 1) and incubated at 37°C in 5% CO₂ for 3 weeks. RNA was harvested from cells, reverse transcribed and semiquantitative PCR was performed using primers complementary to HCMV IE1-72 mRNA and 18S rRNA, as an internal control. Reverse transcriptase negative (RT-) sample was also tested to demonstrate lack of residual, genomic DNA in samples. All experiments were repeated at least three times and representative results are shown.