Growth curves of HCMV in HepG2 cells and PHH.

<p>(A) <i>Growth curves of HCMV in HepG2 cells, PHH and MRC5 cells</i>. HepG2 cells, PHH, and MRC5 cells were left uninfected or infected with HCMV strains AD169 and HCMV-DB (MOI = 1). Inocula were left in place for 2 hours and then removed with three washes of EMEM without serum. Viral titers were determined in the culture supernatants at the indicated times post-infection by real-time PCR. Results are representative of two independent experiments. (B) <i>Viral entry into HepG2 cells, PHH and MRC5 cells</i>. Uninfected cells and cultures infected with HCMV-AD169 at the indicated MOI for 2 hours were treated with trypsin for 10 min and then washed. Samples of extracted DNA were analyzed by PCR using primers specific for the MIEP of HCMV and for beta-globin (internal loading control). The amplification products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. Results are representative of two independent experiments. (C) <i>Detection of IE1 pp72 HCMV antigen in infected HepG2 cells and PHH</i>. HepG2 cells (6×10⁶ cells) and PHH (2×10⁶ cells) were left uninfected or infected with HCMV-AD169 (MOI = 1 and 10). IE1 pp72 HCMV antigen expression was measured at day 3 post-infection by Western blotting as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059591#s2" target="_blank">Materials and Methods</a> section. beta-actin was used as control. Results are representative of two independent experiments. (D) <i>Detection of IE1 pp72, but not US28, pp65 antigen and 65-kD structural late antigen in infected HepG2 cells</i>. HepG2 cells (6×10⁶ cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72, US28, pp65 and 65-kD structural late HCMV antigen expression was measured up to day 4 post-infection by Western blotting as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059591#s2" target="_blank">Materials and Methods</a> section. beta-actin was used as control. Results are representative of two independent experiments. (E) <i>Detection of IE1 pp72 transcript, but not of US28 transcript, in HCMV-infected HepG2 cells</i>. HepG2 cells (6×10⁶ cells) were left uninfected or infected with HCMV-AD169 (MOI = 1). IE1 pp72 and US28 transcript expression was measured up to day 6 post-infection by RT-PCR assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059591#s2" target="_blank">Materials and Methods</a> section. beta-globin was used as control. Results represent means (± SD) of two independent experiments. IE: Immediate Early; MIEP: Major immediate-early promoter; MOI: Multiplicity of infection; PHH: Primary human hepatocytes; U: Uninfected; P: positive control (extract of MRC5 cells infected with HCMV-AD169).