Thr 446 phosphorylation of PKR by HCV core protein deregulates G2/M phase in HCC cells

Abstract

Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma (HCC). HCV core protein affects cell homeostasis, playing an important role in viral pathogenesis of HCC. We investigate the effects of HCV core protein expression on cell growth in HCC cell lines. Cell cycle distribution analysis of HepG2 polyclonal core positive cells reveals a peculiar accumulation of cells in G2/M phase. Different pathways mediate G2/M arrest: such as p53 and double strand RNA protein kinase (PKR). Flow cytometry in p53-null cells demonstrates that p53 plays only a marginal role in inducing HCV core-dependent G2/M phase accumulation that seems to be significantly affected by the functional inactivation of PKR. HCC core positive cells are characterized by a significant PKR phosphorylation in Thr 446 residue, which leads deregulation of mitosis. Moreover, we observe that the overexpression of the viral protein induces an upregulation of PKR activity, which does not correlate with an increased eIF-2 phosphorylation. This uncommon behavior of PKR suggests that its activation by HCV core protein could involve alternative PKR-dependent pathways, implicated in core-dependent G2/M accumulation. The described biological effects of HCV core protein on cell cycle could be an additional viral mechanism for both HCV resistance to interferon (IFN) and HCC HCV-related pathogenesis.

Hepatitis C virus and alcohol: Same mitotic targets but different signaling pathways

“…Moreover, here, we reinforce our previous findings demonstrating that HCV proteins modulate mitotic molecules via PKR [18]. In particular, here we find that the HCV-dependent alterations of cyclin B1, Aurora kinase A, and tyrosine-phosphorylation of gamma-tubulin are mediated by a mechanism strongly dependent on PKR, as well as on p38MAPK and JNK pathways.…”

“…The close association between mitotic defects and hepatocarcinogenesis is also supported by in vivo and vitro studies demonstrating that both HCV and ethanol are able to interfere with the regular control of mitosis in hepatocarcinoma cell lines [18][19][20]. HCV is able to induce an accumulation of cells in G2/M phase and, concomitantly, an increase of cyclin B1/cdk1 complex nuclear translocation, through its effects on p38 mitogen-activated protein kinase (p38MAPK) and RNA-activated protein kinase (PKR), both molecules implicated in the control of G2/ M phase progression [18,19]. Animal and human studies suggest that alcohol may be involved in initiation, promotion, and progression of HCC, through the activation of various molecular mechanisms including oxidative stress, changes in DNA methylation, immunosuppression, and genetic susceptibility [21,22].…”

“…HCV infection, as well as ethanol, impairs cell-cycle progression leading to a G2/M arrest in liver cells [18,20]. Interestingly, over-expression of mitotic molecules, such as cyclin B1 and Aurora kinase A, was found in human HCCs; however, no studies analyzed differences in the expression of these proteins in relation to possible different etiologies of HCCs [34,16,35].…”

“…As already reported, PKR is one mediator of HCV core protein effects on G2/M progression [18,19]. Thus, here, we firstly evaluated whether PKR could be involved in the HCV-related deregulation of cyclin B1, Aurora kinase A, and tyrosinephosphorylated gamma-tubulin.…”

“…After block, G1 cells were released to progress through the cell cycle over the next 15 h, and then treated or not with ethanol. Mitotic index was calculated by DAPI (40-60-diamino-2-phenylindole) staining, as already described [18]. After staining, the slides were extensively washed and mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA), before examination.…”