Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts

Abstract

Replication of human papillomavirus (HPV) genomes requires an origin of replication and two viral proteins: the DNA helicase E1 and the auxiliary factor E2. To dissect the profile of HPV replication in the epithelium, we analyzed replication of an HPV16 origin-containing plasmid in human epithelial cell extracts supplemented with purified E1 and E2. We found that in addition to well-defined circular replication products, high-molecular-weight DNA was synthesized in a manner that depended on the origin, E1 and E2. The high-molecular-weight DNA was converted to a unit-length linear DNA by treatment with restriction enzymes that cleave the plasmid once, implying that a concatemeric DNA was generated by rolling circle replication. Nicking or relaxing the template plasmid enhanced the level of HPV rolling circle replication. In contrast, the addition of an extract from non-epithelial cells diminished the generation of the rolling circle replication product in the epithelial cell extract, indicating factors that counteract HPV rolling circle replication. These results suggest a rolling circle replication mechanism for the HPV genome in cervical epithelial cells, which may have physiological implications for generation of the tandem-repeated HPV genomes occasionally found integrated into the chromosome of cervical cancer cells. IntroductionHuman papillomaviruses (HPVs) are small icosahedral viruses that contain a double-stranded circular DNA genome of approximately 8000 bp (zur Hausen 1996). Among more than 100 types of HPVs so far identified, nearly 15 types are recognized as high-risk types that are closely linked to the development of cervical cancer, with HPV type 16 (HPV16) the predominant high-risk type worldwide (Parkin et al. 2008). HPV infects the basal cells of the epithelium and its genome is maintained as episomal plasmids in the nucleus such that HPV establishes latent infection. When the infected cells leave the basal layer and commence terminal differentiation of the epithelium, the viral genome starts to replicate to yield many thousands of progeny viruses. Thus, replication of the HPV genome is regulated in a strict way that depends on the differentiation status of the host epithelial cells (Longworth & Laimins 2004).The replication of the HPV genome requires an origin of replication and two virally encoded proteins: the DNA helicase E1 and the replication ⁄ transcription factor E2 (Kadaja et al. 2009). To initiate viral DNA replication, E2 binds to a specific binding site at the origin and then recruits E1, leading to the assembly of double E1 hexamers. The resultant E1 hexamer is an active replicative helicase that can induce melting of the DNA at the origin as well as subsequent unwinding of the double helical DNA during replication fork progression. With the exception of continuous DNA unwinding by E1, HPV uses host replication proteins, such as DNA polymerases, proliferating cell nuclear antigen and replication protein A, to accomplish its genome replication (Park et al. 1994;Melendy et al. 1995)....

Molecular mechanisms of HPV mediated neoplastic progression

“…The occasional association of Head-to-tail tandem repeats in the chromosome of cervical cancer cells [58, 59] leads to a plausible explanation that a linear concatemeric HPV genome is synthesized in cells by a rolling circle mechanism of replication and integrates into the host chromosome [60]. Upon induction of differentiation, genome switched to rolling circle mechanism of replication whereas in undifferentiated cells bidirectional replication occurs.…”

Relationship between E6-E7 lineage sequences, viral loads, and integration of HPV16 in women with atypical squamous cells of undetermined significance (ASCUS) pap smears

“…These findings suggest a lower risk of persistence and progression in women with ASCUS Pap smears that were infected with the EUR-350T isolate [8,15,20]. However our technique risks to under-quantify the percentage of integration if the gene E2 is intact after integration, for example in case of tandem-repeated HPV integrated genomes induce by the rolling circle replication mechanism [56,57]. But, HR-HPV DNA is frequently integrated into the chromosome of cervical cancer cells and is occasionally found amplified as tandem-repeated HPV integrated genomes, as typically observed in CaSki cells [21,40].…”

Polyomavirus Large T Antigen Binds Symmetrical Repeats at the Viral Origin in an Asymmetrical Manner

“…However, the asymmetry of the viral origin sequences, combined with the protein asymmetry imposed by steric constraints when the OBDs bind the major groove of the DNA, raises the question of whether all polyomavirus replication proceeds via a bidirectional mechanism. Indeed, rolling-circle replication is well documented for Py (34) and SV40 (35), as well as for papillomavirus 16, which encodes E1, an LT homolog (36). Nonetheless, in these cases, rolling-circle replication is the exception rather than the rule; it is thought to be initiated by a nicked DNA template, a stalled replication fork, or some other perturbation of the normal bidirectional mechanism (35,36).…”

“…Indeed, rolling-circle replication is well documented for Py (34) and SV40 (35), as well as for papillomavirus 16, which encodes E1, an LT homolog (36). Nonetheless, in these cases, rolling-circle replication is the exception rather than the rule; it is thought to be initiated by a nicked DNA template, a stalled replication fork, or some other perturbation of the normal bidirectional mechanism (35,36). The fact that bidirectional replication predominates in vivo in spite of the presumably asymmetrical nature of the LT dodecamer assembly process suggests significant synergy between the two hexamer rings prior to replication initiation.…”