Abstract
ABSTRACTHuman papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.
Highlights
Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection
The minimal origin of replication is sufficient to support the transient replication of viral DNA, but long-term stable maintenance replication of the genomes requires an additional cis element known as the minichromosome maintenance element (MME) [11]
Complementation assay was developed to identify the cis elements required for stable maintenance replication of the HPV18 genome
Summary
Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, curing persistent HPV infection. Viral infections often persist for months and even years [7] This implies that the papillomavirus extrachromosomal genome has to undergo stable maintenance replication in the actively dividing cells within the basal layer. For BPV1, the MME consists of at least six E2 binding sites (E2BS), in addition to those in the replication origin [11]
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