Human Papillomavirus DNA Replication Compartments in a Transient DNA Replication System

Abstract

Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.

HPV31 E7 facilitates replication by activating E2F2 transcription through its interaction with HDACs

“…Recent studies suggest a role for both of these proteins in specific regulation of the Arf promoter and subsequent modulation of cell cycle kinetics (Aslanian et al, 2004). We also observed E2F2 protein to be localized to intranuclear bodies that may coincide with ND10 bodies, which are sites for replication of many DNA tumor viruses, including HPV (Swindle et al, 1999). In support of this idea, E2F2 has been shown to associate with proteins involved in recombinational DNA repair, Mre11 and Nbs1, at both viral and cellular origins of replication (Maser et al, 2001).…”

Prediction of outcome in patients with low‐grade squamous intraepithelial lesions by fluorescence in situ hybridization analysis of human papillomavirus, TERC, and MYC

“…Alternatively, fluorescent dots might not always represent integrated DNA but normally replicating virus DNA. It has been shown that viruses, including HPV, replicate their genome at compartments or foci rather than randomly within the nucleus . Therefore, the HPV FISH probe might hybridize to the accumulated single‐strand DNA of such replication foci, mimicking the punctuate signals noted in the case of integration, thereby explaining the high prevalence of a punctuate pattern (70%) observed in the LSIL cases in the current study.…”

Expression Pattern and Subcellular Localization of Human Papillomavirus Minor Capsid Protein L2

“…the viral origin. 9,35 However, E1 and E2 co-expressed in C33A cells harboring a plasmid with a HPV origin of replication do indeed co-localize with PML. 35 Furthermore, they co-localize with RP-A and BrdU staining indicating active sites of replication.…”

“…9,35 However, E1 and E2 co-expressed in C33A cells harboring a plasmid with a HPV origin of replication do indeed co-localize with PML. 35 Furthermore, they co-localize with RP-A and BrdU staining indicating active sites of replication. 35 However, for papillomavirus, neither the E2-dependent transcription nor viral DNA replication is reliant on the presence of PML.…”

“…35 Furthermore, they co-localize with RP-A and BrdU staining indicating active sites of replication. 35 However, for papillomavirus, neither the E2-dependent transcription nor viral DNA replication is reliant on the presence of PML. 12 Notably, during initial papillomavirus infection, it is L2, not L1, that accompanies the viral genome into the nucleus and trafficks to ND-10.…”