Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses

Abstract

In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection.

Biology of Human Papillomavirus Infections in Head and Neck Carcinogenesis

“…In early carcinogenesis, the concomitant presence of both circular HPV genomes and integrated form in the same cells might be the crucial event for additional chromosomal changes needed to acquire a growth advantage as supported by our in vitro model [4]. Thus, in early HPV infection, if both episomal and integrated forms of HPV exist in the same cell, the replication of integrated HPV leads to rearrangements within the integrated locus being thus the key event in the ''hit-and-run'' mechanisms [55]. The HPV biology of head and neck cancer has also recently been discussed in two excellent reviews [56,57].…”

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…High local CRC, historically known as the "onion-skin DNA replication," is observed in eukaryotic cells at sites of chromosomal integration of papillomavirus and poliomavirus genomes (Syu and Fluck 1997;Kadaja et al 2009), as well as at "puffs" of developmentally regulated local chromosome amplifications in insects (Spradling 1981;Liang et al 1993). There is also a possibility that stable elevated replication complexity explains certain cases of copy number variation in cells of higher eukaryotes and the origin of certain chromosome rearrangement events (Hastings et al 2009;Zhang et al 2009).…”

The Role of E2 Proteins in Papillomavirus DNA Replication

“…The replication forks initiated at the integrated HPV origins extend into the flanking regions of cellular DNA, and these amplified genomic sequences could be targets for the recombination and repair system. This suggests that replication induced from the papillomavirus integrated origin may induce genomic changes of the host cell (Kadaja et al, 2009;Kadaja et al, 2007). Fig.…”