Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

Abstract

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPVinfected patients. Human papillomaviruses (HPV) are important pathogens that cause different epithelial hyperplastic lesions, most commonly manifesting as benign warts or papillomas. Over 100 HPV types have been identified to date (1). These epitheliotropic viruses can be categorized based on their ability to infect mucosal or cutaneous keratinocytes. The mucosal viruses can be further subdivided into low-and high-risk HPVs. A potential for malignant progression is characteristic of high-risk HPV types, such as HPV18, HPV16, HPV31, and HPV45, whereas types such as HPV6 and HPV11 do not show similar associations and are considered low risk (2). Essentially all cervical carcinomas (3) and a quarter of reported head and neck cancers (4) are associated with HPV infections.HPV is a small DNA virus with an approximately 8-kbp genome. During infection of stratified cutaneous or mucosal epithelia, the viral genomes replicate as multicopy extrachromosomal genetic elements in the nuclei of host cells. HPV genomes undergo a three-phase replication cycle linked to the host cell differentiation program (5). The first stage of HPV DNA replication occurs in undifferentiated basal keratinocytes after infection and is referred to as transient amplificational replication. During the first phase, the viral replication factors are produced and the HPV genome is amplified up to 100 of copies per cell during the S phase of the cell cycle. After initial amplification, the expression of viral replication proteins is downregulated to a level sufficient for the stable maintenance phase of episomal genomes in HPV-infected basal cells. Upon differentiation of the infected cells, the regulated expression of viral proteins initiates a second amplification of the viral genome, the production of capsid proteins, and the assembly of viral particles in the uppermost layers of terminally differentiated epithelium. The mechanisms regulating the switch from the initial HPV genome replication to HPV genome maintenance and, subsequently, to vegetative amplification are not entirely understood.Replication of the HPV genome is carried out by the cellular replication machinery, which is directed to the viral origin by the viral replication proteins E1 and E2. The mechanism underlying the initiation of DNA replication is well described...

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

“…1A. With D/H-digested DNA, a pattern of migration was observed for HPV18 sequences that was consistent with open circular and supercoiled forms of episomal DNA (48). Digestion with EcoRI resulted in all hybridizing species migrating at the position of the ϳ8-kb linearized nonintegrated HPV genome as the linearized pBluescript-HPV18 plasmid digested with EcoRI (Fig.…”

Could the human papillomavirus vaccines drive virulence evolution?

“…Furthermore, following several challenges to the prevailing view of slow dsDNA virus evolution (where mechanisms such as recombination are possible [57][58][59][60][61]), there is a need for more direct investigations into the evolutionary potential of HPV variants.…”

Papillomavirus Replication

“…Investigators working with HPV-positive cancer cell lines initially believed that viral genomes in these cells only existed in an integrated form, though Choo et al (1989) discovered episomes as interlocking rings of HPV16 dimers. In transfected U2OS and C33A cells, HPV-positive SiHa and HeLa cells, and cervical and oropharyngeal tumors, Orav et al (2013) also found catenated (chain-linked) multimeric episomes. A recent study of cervical cancer samples reported a range of PV genome presentations, from one integrated copy to integrated tandem repeats to coexisting integrated and episomal forms.…”

“…For instance, in ID13 cells, a C127-derived cell line, BPV genomes were found not only as monomeric and oligomeric episomes but were also found in tandem repeats integrated within host chromosomes (Schvartzman et al 1990). Even though PV genomes are typically maintained as monomeric, multi-copy episomes, some studies have reported the presence of multimeric episomes both in cell culture and in samples from infected patients (Kennedy et al 1987, Choo et al 1989, Kristiansen et al 1994, Orav et al 2013.…”

“…In an in vitro assay, nicking DNA facilitated rolling-circle replication (Kusumoto-Matsuo et al 2011). Although episomal multimers are cited as evidence for this mode, HR may also generate multimers (Orav et al 2013); it is unclear which mechanism is used in vivo since either nicks (favoring the former) or dsDNA breaks (promoting the latter) could occur. Studies demonstrate co-localization of Rad51, Rad52, yH2AX, and pNbs1 with HPV replication foci along with ATM activation, suggesting the involvement of dsDNA breaks in some cases of PV replication (Gillespie et al 2012, Sakakibara et al 2013b.…”