HBx Protein of Hepatitis B Virus Promotes Reinitiation of DNA Replication by Regulating Expression and Intracellular Stability of Replication Licensing Factor CDC6

Vijaya Pandey,Vijay Kumar

doi:10.1074/jbc.m112.359760

Abstract

Prevention of re-replication via negative regulation of replication initiator proteins, such as CDC6, is key to maintenance of genomic integrity, whereas their up-regulation is generally associated with perturbation in cell cycle, genomic instability, and potentially, tumorigenesis. The HBx oncoprotein of hepatitis B virus is well known to deregulate cell cycle and has been intricately linked to development of hepatocellular carcinoma. Despite a clear understanding of the proliferative effects of HBx on cell cycle, a mechanistic link between HBx-mediated hepatocarcinogenesis and host cell DNA replication remains poorly perused. Here we show that HBx overexpression in both the cellular as well as the transgenic environment resulted in the accumulation of CDC6 through transcriptional and post-translational up-regulation. The HBx-mediated increase in CDK2 activity altered the E2F1-Rb (retinoblastoma) balance, which favored CDC6 gene expression by E2F1. Besides, HBx impaired the APC(Cdh1)-dependent protein degradation pathway and conferred intracellular stability to CDC6 protein. Increase in CDC6 levels correlated with increase in CDC6 occupancy on the β-globin origin of replication, suggesting increment in origin licensing and re-replication. In conclusion, our findings strongly suggest a novel role for CDC6 in abetting the oncogenic sabotage carried out by HBx and support the paradigm that pre-replicative complex proteins have a role in oncogenic transformation.

Highlights

CDC6 is a replication licensing factor, which is tightly regulated to prevent reinitiation of replication
HBx Regulates Stability of Origin Licensing Factor CDC6—As deregulation of CDC6 is frequently seen in many human cancers (19 –22), its involvement in hepatocellular carcinoma was investigated under the HBx microenvironment
There was a progressive increase in CDC6 protein levels in the presence of HBx, but it remained unaffected in the case of control and mutant X9 (Fig. 1A)

Summary

Background

CDC6 is a replication licensing factor, which is tightly regulated to prevent reinitiation of replication. Results: Hepatitis B virus HBx protein up-regulates CDC6 both transcriptionally as well as post-transcriptionally and promotes its recruitment to ␤-globin origin of replication. In early G1 phase of the cell cycle, CDC6, along with Cdt (chromatin licensing and DNA replication factor 1), forms a pre-replication complex with a six-subunit origin recognition complex on the origins of replication followed by recruitment of the minichromosome maintenance complex 2–7 helicases. These molecular events lead to licensing of origins, from which DNA synthesis begins at the onset of S-phase [6, 7]. We show that increased CDC6 levels correlated with its specific enrichment on the ␤-globin origin of replication that seems to favor origin licensing, re-replication, and unscheduled entry of cells into S-phase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION