HeLa Cells Are Phenotypically Limiting in Cyclin E/CDK2 for Efficient Human Papillomavirus DNA Replication

Abstract

Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/ cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Small DNA viruses such as SV40/polyomavirus and papillomaviruses each encode one or two viral origin (ori) recognition and initiator proteins and rely heavily on the cellular DNA replication machinery. Because the interactions among viral and cellular proteins mimic those among the cellular counterparts, these viruses have proven to be particularly useful model systems. The SV40 system has aided in the identification and mechanistic description of several important replication components (reviewed in Ref.2) and more recently has revealed the opposing effects of cyclin E/CDK2 and cyclin A/CDK2 in regulating DNA polymerase ␣/primase via direct phosphorylation (3). The fact that papillomaviruses are highly prevalent and medically important human and animal pathogens also makes them attractive to investigators (reviewed in Ref. 4).The genome of papillomaviruses is an approximately 8-kilobase pair double-stranded circular DNA. Because the productive life cycle takes place only in keratinocytes undergoing terminal squamous differentiation (4), viral DNA replication is studied either in a cell-free system or in transiently transfected cells (reviewed in Refs. 5 and 6). The ori of all papillomaviruses consists of one binding site for the virus-encoded E1 protein and several highly conserved binding sites for the virus-encoded E2 protein. E2 is a critical origin recognition protein (7-15), and E1 is the initiator and a DNA helicase (16 -20). They are functionally equivalent to the cellular origin recognition complex and MCM complexes, respectively, during initiation. Replication is dependent on the cellular DNA replication machinery, including DNA polymerase ␣/primase, DNA polymerase ␦, replication protein A (RPA, the single-stranded DNAbinding protein), the proliferating cell nuclear antigen (the processivity clamp for DNA polymerase ␦), replication factor C (the clamp loader), and topoisomerases I and II (8,14). As a consequence of this high degree of conservation in cis sequence elements and trans-acting factors, E1 and E2 proteins from any one virus can replicate all papillomavirus origins (Refs. 21 and 22 and references therein).E1 protein interacts directly with the p180 and p70 subunits of DNA polymerase ␣/primase (23-26) and with RPA (27) and is required throughout the initiation and elongation stages, whereas E2 protein is nee...

The Hinge of the Human Papillomavirus Type 11 E2 Protein Contains Major Determinants for Nuclear Localization and Nuclear Matrix Association

“…HPV-11 E2 protein is a substrate of cyclin E-, A-, or B-dependent kinases (CDKs) in vitro in a complex which also contains the HPV-11 E1 protein (41). However, only cyclin E/cdk2 plays a unique role in initiation of replication that cannot be fulfilled by other CDKs (38). We note that several candidate CDK phosphorylation sites are located in the hinge region.…”

Alternative Fates of Keratinocytes Transduced by Human Papillomavirus Type 18 E7 during Squamous Differentiation

“…Interestingly, the papillomavirus-encoded DNA helicase E1 and the primary origin recognition protein E2 are substrates of various cdks in vitro (9,24). In particular, phosphorylation of E1 by cyclin E/cdk2 is critical for efficient initiation of HPV DNA replication (23).…”

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

“…However, a previous study using W12 cells, which were isolated from cervical intraepithelial lesion and retain HPV16 genomes as episomes (Stanley et al 1989), reported that replication of the HPV16 genome switches from a bidirectional to a unidirectional mode on cell differentiation, which implies a rolling circle mode of HPV replication (Flores & Lambert 1997). As an extract from HeLa cells was examined for its ability to support HPV replication without success (Lin et al 2000), in vitro HPV replication has not been studied using extracts from human epithelial cells.Here, to examine how HPV replication progresses in epithelial cells, we have employed an in vitro replication assay using crude extracts from differentiated epithelial cells. In addition to bidirectional replication, we have demonstrated that an extract from W12 cells induces a substantial level of rolling circle replication of an HPV16 origin-containing plasmid in a manner that depends on the presence of E1 and E2.…”

“…However, a previous study using W12 cells, which were isolated from cervical intraepithelial lesion and retain HPV16 genomes as episomes (Stanley et al 1989), reported that replication of the HPV16 genome switches from a bidirectional to a unidirectional mode on cell differentiation, which implies a rolling circle mode of HPV replication (Flores & Lambert 1997). As an extract from HeLa cells was examined for its ability to support HPV replication without success (Lin et al 2000), in vitro HPV replication has not been studied using extracts from human epithelial cells.…”