2C). To assess the extent of HCMV inactivation selleck chemicals 17-AAG by UV treatment, we infected MRC-5 with UV-treated virus. We observed that UV-treatment almost completely abolished virus infectivity and IE1 expression (Fig. 2D). Taken together, these data suggest that the induction of IL-6 was at least in part dependent on viral replication cycle (probably expression of IE HCMV proteins) in HCMV-infected HepG2 cells and PHH. Figure 2 HCMV induces secretion of IL-6 by HepG2 cells and PHH. HCMV induces IL-6-mediated JAK-STAT3 activation in HepG2 cells and PHH IL-6 binds to the IL-6 receptor (IL-6R) to activate STAT3 signaling [6]. Therefore we assessed the phosphorylation status of STAT3 in HepG2 cells and PHH infected with HCMV.

Consistent with the presence of IL-6 in the supernatant, STAT3 phosphorylation was markedly increased in HepG2 cells and PHH infected with HCMV compared to mock-infected cells (Fig. 3A). In HepG2 cells, STAT3 phosphorylation was detected as early as 2 h post-infection, peaked 1 day post-infection, and decreased thereafter (Fig. 3A). In contrast, STAT3 phosphorylation was detected as early as 2 h post-infection in PHH and peaked again at day 3 post-infection (Fig. 3A). Both HCMV-AD169 and HCMV-DB strains activated STAT3 in HepG2 cells and PHH (Fig. 3A). In contrast to infection with UV-HCMV, ganciclovir pretreatment of the cells did not prevent STAT3 activation in PHH infected with HCMV (Fig. 3A and 3D), indicating that STAT3 activation, like IL-6 production, did require early steps of viral replication. Figure 3 HCMV induces IL-6-mediated activation of the JAK-STAT3 axis in HepG2 cells and PHH.

Since cytokine activation of STAT3 is mediated by upstream Janus kinases (JAKs) [6], we assessed the expression of JAK-1 and JAK-2 in HepG2 cells and PHH infected with HCMV. JAK-1 and/or JAK-2 activation was increased in HepG2 cells and PHH infected with AD169 or HCMV-DB compared to mock-infected cells (Fig. 3B). Pretreatment of HCMV-infected HepG2 cells and PHH with a pan-JAK inhibitor and a STAT3 inhibitor greatly reduced STAT3 phosphorylation (Fig. 3C), indicating activation of a JAK-STAT3 axis in HepG2 cells and PHH infected with HCMV. Since the binding of IL-6 to IL-6R activates STAT3, we directly assessed the role of IL-6R in STAT3 activation in HepG2 cells and PHH.

HCMV infection induced STAT3 activation in both cell types, whereas incubation of HCMV-infected cells with an IL-6R neutralizing antibody decreased STAT3 phosphorylation (Fig. 3C). In contrast, incubation with an EGF receptor (EGFR) neutralizing antibody did not inhibit STAT3 activation by HCMV in HepG2 cells (Fig. 3C). Moreover, incubation of cells with the recombinant glycoprotein gB, which was previously shown to bind to and activate EGFR-mediated pathways [28], failed to activate STAT3 (Fig. 3C). In contrast to infection with Brefeldin_A live HCMV, decreased activation of STAT3 and JAK2 was observed in cells treated with UV-inactivated HCMV (Fig. 3D).