We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tightbinding substrate of cyclin E͞cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1͞E2 complex, and A-and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin͞Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replicationinitiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.Cell cycle transitions are coordinated in large part through the action of a family of cyclin-dependent kinases (Cdks), enzymes composed of a catalytic Cdk subunit, and a regulatory cyclin subunit (1). Much of what we know concerning Cdk action in the G 1 ͞S transition comes from analysis of the Rb͞E2F pathway, which has led to the identification of Rb, p107, p130, E2F-1, and DP-1 as Cdk substrates (1, 2). Phosphorylation of these proteins typically is linked with formation of tight complexes between the kinase and the substrate, most frequently through a motif in the substrate (the RXL motif) that interacts with the cyclin box. The RXL motif forms the basis for interaction of the p21 family of Cdk inhibitors with cyclins (3-6) and is important for Cdk-mediated phosphorylation of p107 (6, 7), E2F-1 (6, 8, 9), and Rb (10). Cyclin E͞Cdk2 is a key regulator of S-phase initiation. Removal of cyclin E͞Cdk2 activity from Xenopus egg replication systems (reviewed in ref. 11) or mammalian cells (12) blocks S-phase entry, and inXenopus, this blockade can be overcome by addition of cyclin E͞Cdk2. Conversely, ectopic expression of cyclin E can initiate replication independent of Rb inactivation in mammalian cells and in Drosophila (ref. 13, and reviewed in ref. 2). However, targets of this kinase in the preinitiation complex are unknown. We and others (14) have taken advantage of the tight association of cyclins with their substrates to identify cyclin E͞Cdk-binding proteins and substrates via expression cloning techniques. Here we report the identification of human papillomavirus (HPV) replication-initiation protein E1 as a tightbinding substrate of cyclin͞Cdk complexes.Papill...

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.Precise and timely subcellular localization of proteins is essential for their biological functions, and conversely, the control of protein localization provides the cells with a convenient way to regulate their functions. Posttranslational modifications of proteins play critical roles in these aspects. For instance, three of the most important outcomes after protein phosphorylation are typically the changes in protein localization, stability, or activity (32, 38). Eukaryotic DNA replication is strictly controlled by mechanisms that regulate the cell cycle to ensure both genetic inheritance and stability. DNA replication is initiated at a precise time when the cellular replication machinery is ready, and then the genome is replicated for a single round in each cell cycle. Cell cycle entry and progression are regulated by cyclin/cyclin-dependent kinases (CDKs) that phosphorylate key regulatory proteins (5,59,65,74). At the G 1 /Sphase transition, cyclin/CDK complexes phosphorylate the components of the cellular DNA prereplication complex (pre-RC), including origin recognition complex, Cdc6, Cdt1, and MCM2-7, enabling the initiation of replication and subsequently preventing reduplication in the same cell cycle (5, 6, 54). The specific mechanisms vary for different components in different organisms. For example, for the replicative DNA helicase complex composed of the MCM2-7 subunits (71), phosphorylation inhibits its activity in humans and mice (29,30). In budding yeast, phosphorylation leads t...

Human papillomaviruses (HPVs) establish long-term infections in patients. The mechanism for extrachromosomal HPV DNA persistence in cycling cells is unknown. We show that HPV origincontaining plasmids partition as minichromosomes, attributable to an association of the viral origin recognition protein E2 with mitotic spindles. ␣-, ␤-, and ␥-tubulins were pulled down with a tagged E2. The N-terminal transacting and C-terminal protein dimerization͞DNA binding domains independently associated with the spindles. We suggest that this E2 property enables these viruses to establish persistence. Its implication for HPV oncogenesis is presented.F or any extrachromosomal DNA virus to establish a persistent infection in cycling host cells, the viral genome must replicate and partition into both daughter cells during division. The E2 origin (ori)-binding protein of bovine papillomavirus type 1 (BPV-1) associates with mitotic chromosomes (1-3), thus providing a mechanism for viral DNA segregation. Comparable mechanisms have been demonstrated for the Epstein-Barr virus through the Epstein-Barr virus-encoded nuclear antigen 1 protein and the Kaposi's Sarcoma virus (human herpesvirus 8) through the latency-associated nuclear antigen 1 protein (4, 5). In contrast, the mechanism by which human papillomavirus (HPV) DNA partitions during cell division has not been elucidated. In this report, we demonstrate that HPV ori-containing DNA segregates as minichromosomes by association with mitotic spindles and this association is mediated by the HPV origin recognition protein E2.HPVs are medically important pathogens that establish persistent infections in long-living basal keratinocytes. Infections typically cause benign hyperproliferation of squamous epithelia in the form of cutaneous warts, laryngeal papillomas, and anogenital condylomata. Over time, infections can become subclinical, but may reactivate during episodes of immune suppression. The HPV genome is a double-stranded, circular DNA of Ϸ7,900 bp and replicates extrachromosomally in the nucleus of infected keratinocytes. Low copy numbers of the mucosotrophic HPV DNA plasmids are maintained in the basal and parabasal cells that divide, whereas the productive phase takes place only in postmitotic, differentiated cell strata and progeny virus shed within the sloughing superficial cells (6). Thus, it is paramount that, in either latent or active infections, HPV DNA must partition into the two daughters of dividing cells for viral persistence. To support viral DNA amplification in postmitotic cells, the viral E6 and E7 proteins inactivate the host tumor suppressor proteins p53 and pRB (retinoblastoma protein), respectively, reestablishing an S-phase environment. Elevated transcription of these oncogenes is normally limited to the differentiated compartment. However, if inappropriately expressed in the basal cells, such as during repeated wounding and healing, the high-risk HPV oncoproteins can promote excessive cell cycling and host chromosome instability. Indeed, a small fraction of ...