Abstract
Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against HPV infection. We used two-dimensional and three-dimensional gel electrophoresis techniques to identify replication intermediates arising during the initial amplification of HPV18 episomal genomes. We determined that the first rounds of HPV18 replication proceed via bidirectional theta structures; however, a notable accumulation of almost fully replicated HPV18 genomes indicates difficulties with the completion of theta replication. We also observed intermediates that were created by a second replication mechanism during the initial amplification of HPV18 genomes. The second replication mechanism does not utilize specific initiation or termination sequences and proceeds via a unidirectional replication fork. We suggest a significant role for the second replication mechanism during the initial replication of the HPV18 genome and propose that the second replication mechanism is recombination-dependent replication.
Highlights
Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against Human papillomaviruses (HPVs) infection
2D N/N agarose gel electrophoresis (AGE) analysis of uncut replication intermediates (RIs) created during the initial replication of episomal HPV18 genomes in U2OS cells
We have previously demonstrated the increasing prevalence of oligomeric HPV genomes in HPV-transfected U2OS cells in time[19,26] and characterized these oligomers as episomal head-to-tail concatemeric molecules, which likely arise from monomeric HPV genomes through replication-dependent homologous recombination[26]
Summary
Determining the mechanism of HPV18 replication is paramount for identifying possible drug targets against HPV infection. We used two-dimensional and three-dimensional gel electrophoresis techniques to identify replication intermediates arising during the initial amplification of HPV18 episomal genomes. The initial amplification phase is followed by a stable maintenance of the viral episomes It is unknown which replication mechanism is behind the transient amplification phase; the stable maintenance replication of papillomavirus genomes proceeds via bidirectional theta structures[16,17]. We used the U2OS-based model system[19] to study replication intermediates (RIs) that arise during the initial amplification of the wild-type HPV18 (HPV18wt) and HPV18E8− mutant genomes. We analyzed RIs that arose during the initial amplification of HPV18E8− mutant genomes in HaCaT cells and found them to be similar to the RIs that arose during the initial amplification of HPV18 genomes in the U2OS cell line
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