Up-regulation of cyclin D1 and survivin in HepG2 cells and PHH infected with HCMV.

<p>(A) <i>Time course of the expression of cyclin-D1 and survivin in HepG2 cells infected with HCMV.</i> HepG2 cells (6×10⁶ cells) were left uninfected or infected with HCMV strains AD169 (MOI = 0.5) and HCMV-DB (MOI = 1.0). Cyclin D1 and survivin expression was measured 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>, and beta-actin was used as an internal control. (B) <i>Expression of cyclin-D1 and survivin in PHH and HepG2 cells infected with live HCMV or UV-inactivated HCMV</i>. HepG2 cells (6×10⁶ cells) and PHH (2×10⁶ cells) were left uninfected or infected with HCMV or UV-inactivated HCMV (AD169, MOI = 0.5). Cyclin D1 and survivin expression was measured 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>, and beta-actin was used as an internal control. (C) <i>Expression of cyclin D1 and survivin is mediated primarily by HCMV in HepG2 cells and PHH.</i> The histogram shows survivin and cyclin D1 expression at day 3 post-infection as quantified using Image J 1.40 software. Results of western-blots are representative of two independent experiments; histogram represents means (± SD) of two independent experiments.</p

HCMV upregulates p53 and p21 in HepG2 cells and PHH.

<p>Time course of p53, p21 and Mdm2 in HepG2 cells and PHH infected with HCMV. HepG2 cells (6×10⁶ cells) and PHH (2×10⁶ cells) were left uninfected or infected with HCMV strains AD169 and HCMV-DB at MOI = 0.5 and 1, respectively. P53, p21 and Mdm2 protein expressions were measured by Western blotting, and beta-actin was used as an internal control. Results are representative of two independent experiments.</p

Potential oncogenic effect of the IL-6-JAK-STAT3 axis in PHH.

<p>Potential oncogenic effect of the IL-6-JAK-STAT3 axis in PHH.</p

Measurement of NF-κB activation in PBMCs, PBLs and monocytes isolated from the peripheral blood of HIV-HCV coinfected patients and HCV monoinfected patients.

<p>(A) NF-κB activation measured by EMSA in PBMCs, PBLs and monocytes isolated from the peripheral blood of a coinfected patient and a monoinfected patient. Results are representative of twelve independent experiments (7 coinfected patients, 5 monoinfected patients). (B) Mean values (± S.D.) of NF-κB activation in PBMCs, PBLs and peripheral blood monocytes isolated from coinfected patients and monoinfected patients, using an EMSA followed by quantification with a phosphoimager as described in Materials and Methods. *p≤0.05.</p

Plasma HCV loads in HIV-HCV coinfected patients and in HCV monoinfected subjects.

<p>Individual values (upper panel) and mean values (±S.D.) (lower panel) of plasma HCV RNA loads were measured. p = NS.</p

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).</p

HCMV infection increases HepG2 tumorsphere formation.

<p><i>Upper panel:</i> HepG2 cells and MRC-5 cells were infected with HCMV, UV-inactivated HCMV or left uninfected. The tumorsphere formation was assessed at day 9–10 post-infection. Representative phase contrast micrographs of HepG2 tumorspheres are shown. As a negative control, HCMV-infected MRC5 cells did not form tumorspheres. Magnification: 100× (200× in upper left corner of each picture). <i>Lower panel:</i> When we challenged the HepG2 cultures to form tumorspheres, we found that HCMV infection formed 2.5-fold more tumorspheres than uninfected cultures. The histogram presents tumorsphere formation presented as the average number of spheres per 10,000 cells plated (± SEM). Results represent three biological replicates. **P<0.01.</p

Characteristics of the population studied.

<p>n/a, not available.</p

Preferential detection of intracellular HCV in peripheral blood monocytes of HIV-HCV coinfected patients.

<p>Means (±S.D.) of intracellular HCV loads measured in autologous PBMCs, PBLs and monocytes isolated from the peripheral blood of HCV monoinfected and HIV-HCV coinfected patients as described in Materials and Methods are indicated. *p≤0.05.</p

HCMV triggers cell proliferation via IL-6-STAT3 activation in HepG2 cells and PHH.

<p>HepG2 cells (6×10⁶ cells) and PHH (2×10⁶ cells) were left uninfected or infected with AD169 (MOI = 0.5) and UV-inactivated HCMV. Cell proliferation was measured by monitoring the expression of Ki-67 Ag using flow cytometry and MTT 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>. (A) Ki67 Ag expression as measured by flow cytometry. MFI, mean fluorescence intensity. Results are representative of two independent experiments. (B) Histograms show Ki-67 Ag and MTT data from two independent experiments. (C) Increased cell proliferation in HepG2 cells infected with AD169 is blocked by a neutralizing anti-IL-6R mAb (10 microg/ml), a Jak inhibitor (1 micromol/l) and a STAT3 inhibitor (10 micromol/l), and decreased cell proliferation was observed in HepG2 cells infected with UV-inactivated HCMV. Mean values ± SD are representative of two independent experiments.</p