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 ...

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, singlestranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.Human and animal papillomaviruses are prevalent pathogens. Efficient origin (ori)-dependent replication of viral DNA requires the virus-encoded E1 and E2 proteins as well as cellular replication proteins (11,27,46,59,68). As such, these DNA viruses may serve as a model for higher eukaryotic DNA replication, as do simian virus 40 (SV40) and polyomavirus. The papillomavirus ori consists of several E2 binding sites (BS) flanking one E1 BS. E1 recruits the DNA polymerase ␣/primase (6, 12, 41) and the single-stranded-DNA-binding protein RPA (25). The human papillomavirus (HPV) E1 protein is required during initiation and elongation and is thought to be the replicative helicase (33). However, HPV E1 proteins are poor helicases in strand displacement assays (26, 65), and there has been no report of DNA-unwinding activity. In contrast, the bovine papillomavirus 1 (BPV-1) E1 exhibits helicase activity in both assays (51, 69).We previously reported that purified HPV-11 E1 protein expressed in insect Sf9 cells binds to ori with low affinity and specificity and also binds to DNA nonspecifically (33). Electron microscopy (EM) shows that E1 binds ori primarily as a hexamer and, at a low frequency, as a dihexamer (34). The human heat shock proteins Hsp70, Hdj2, and Hdj1 greatly stimulate E1 binding to ori. Hdj1 and Hdj2 encode members of the Hsp40 family of proteins that normally function as cochaperones of the Hsp70 proteins and greatly stimulate the ATPase activity of Hsp70 (for a review, see reference 21). However, in the case of the HPV-11 E1-ori association, their effects are independent and additive. Most strikingly, EM has revealed that Hsp40 but not Hsp70 promotes E1 dihexamer formation on ori (34). The BPV-1 E1 has been reported to be a hexameric helicase (48). However, EM revealed a bilobed comp...

Human and animal papillomavirus DNA replicates as multicopy nuclear plasmids. Replication requires two viral proteins, the origin-recognition protein E2 and the replicative DNA helicase E1. Using genetic, biochemical, and immunofluorescence assays, we demonstrated that efficient nuclear import of the human papillomavirus (HPV) type 11 E1 protein depends on a codominant bipartite nuclear localization sequence (NLS) and on phosphorylation of the serine residues S89 and S93 by the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase, and c-Jun N-terminal protein kinase. The NLS and the MAPK substrates are located within a 50-amino-acid-long peptide near the amino terminus, previously designated the localization regulatory region (LRR). The downstream NLS overlaps the cyclin-binding motif RRL, which is necessary for phosphorylation by the cyclin-dependent kinases to inactivate a dominant nuclear export sequence, also in the LRR. Alanine mutations of the MAPK substrates significantly impaired nuclear import, whereas phosphomimetic mutations partially restored nuclear import. We further identified two MAPK docking motifs near the C terminus of E1 that are conserved among E1 proteins of many HPVs and bovine papillomavirus type 1. Mutations of these MAPK docking motifs or addition of specific MAPK inhibitors significantly reduced nuclear import. Interestingly, a fraction of the NLS-minus E1 protein was cotransported with the E2 protein into the nucleus and supported transient viral DNA replication. In contrast, E1 proteins mutated in the MAPK docking motifs were completely inactive in transient replication, an indication that additional properties were adversely affected by those changes.Infections by human papillomaviruses (HPVs) can cause benign, hyperproliferative lesions of cutaneous or mucosal epithelium. The virus has a double-stranded circular DNA genome approximately 7,900 bp in length, which replicates as extrachromosomal nuclear plasmids. A low copy number of the viral DNA is maintained in the cycling basal and parabasal keratinocytes of squamous epithelium. Viral DNA amplification to produce progeny virions occurs only in postmitotic, suprabasal cells undergoing terminal differentiation (for a review, see reference 15). Initiation of replication from the origin (ori) of various HPV genotypes and bovine papillomavirus type 1 (BPV-1) depends on the virus-encoded ori binding protein E2 and the replicative DNA helicase E1 (for reviews, see references 16 and 63). The ori consists of several E2 protein binding sites flanking a cluster of E1 protein binding sites. The structures and functions of the E1 and E2 proteins of human and animal papillomaviruses are largely conserved, but significant differences are also noted. In brief, the 42-kDa E2 protein binds as dimers to the palindromic ori sequences, ACCGNNNNCGGT, and recruits the 70-kDa E1 protein via an interaction between the carboxyl terminus of E1 and the amino terminus of E2 (16). E1 then assembles into a dihexameric helicase (28,44...

The E2 protein of papillomaviruses is a site-specific DNA binding nuclear protein. It functions as the primary replication origin recognition protein and assists in the assembly of the preinitiation complex. It also helps regulate transcription from the native viral promoter. The E2 protein consists of an amino-terminal (N) trans-acting domain, a central hinge (H) domain, and a carboxyl-terminal (C) protein dimerization and DNA binding domain. The hinge is highly divergent among papillomaviruses, and little is known about its functions. We fused the enhanced green fluorescent protein (GFP) with the full-length human papillomavirus type 11 (HPV-11) E2 protein and showed that the resultant fusion, called gfpE2, maintained transcription and replication functions of the wild-type protein and formed similar subnuclear foci. Using a series of GFP fusion proteins, we showed that the hinge conferred strong nuclear localization, whereas the N or C domain was present in both cytoplasm and nucleus. Biochemical fractionation demonstrated that the N domain and hinge, but not the C domain, independently associated with the nuclear matrix. Mutational analyses showed that a cluster of basic amino acid residues, which is conserved among many mucosotropic papillomaviruses, was required for efficient nuclear localization and nuclear matrix association. This mutation no longer repressed the HPV-11 upstream regulatory region-controlled reporter expression. However, a very small fraction of this mutant colocalized with E1 in the nucleus, perhaps by a piggyback mechanism, and was able to support transient replication. We propose that the hinge is critical for the diverse regulatory functions of the HPV-11 E2 protein during mRNA transcription and viral DNA replication.The papillomavirus E2 protein is a multifunctional nuclear phosphoprotein with a molecular mass of approximately 40 kDa. With high affinity and specificity, it functions as a dimer, binding to multiple copies of a consensus E2 binding site (E2BS), ACCN 6 GGT, located in the upstream regulatory region (URR) and regulating both viral DNA replication and mRNA transcription from the viral E6 promoter located immediately upstream of the E6 gene. The E2 protein is the primary origin recognition protein and helps recruit the viral replication initiator E1 protein (8,30,40,55,64,66,70). It also plays a role in the assembly of the preinitiation complex (39; K.-Y. Lee, T. R. Broker, and L. T. Chow, unpublished data). In vitro, E2 is able to exclude nucleosome formation on the origin, which would otherwise inhibit the initiation of DNA replication (36). Furthermore, E2 modulates the native human papillomavirus (HPV) URR-E6 promoter or a surrogate promoter linked to the URR or to one or more copies of synthetic E2BS (16-18, 26, 59, 62, 63). The bovine papillomavirus type 1 (BPV-1) E2 protein tethers the BPV-1 DNA to mitotic chromosomes, ensuring plasmid segregation during mitosis (28,35,58). Moreover, the BPV-1 E2 has been postulated to play a role in virion morphogenesis (14).The fu...

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 human papillomavirus (HPV) origin (ori) of DNA replication shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundred base pairs. The HPV type-11 ori spans 103 bp and contains three palindromic binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 origin binding protein. These sites are separated by 64 bp and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 ori DNA bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies of the dependence of looping on salt, ATP, and DTT using full length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N terminal domain as well as factors which may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed.