Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase

125

194

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high-and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G 2 phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of ␥H2AX, ATR-interacting protein (ATRIP), and topoisomerase II␤-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification. P apillomaviruses are species-specific double-stranded DNA (dsDNA) viruses that infect the cutaneous and mucosal epithelia of many vertebrate species (1). Human papillomavirus (HPV) infections are widespread, and this virus is considered a common member of the human epithelial microflora (2). In many cases, infections with papillomaviruses are asymptomatic (3). Nearly 100 different HPV types have been identified (4); infections with low-risk viruses (e.g., HPV type 6b [HPV6b] and HPV11) might induce the formation of benign tumors, such as warts and condylomas, while other types (e.g., HPV16 and HPV18), which are referred to as high-risk types, have been shown to cause anogenital and head and neck cancers (reviewed in reference 5). The viral genomes are maintained in infected cells as extrachromosomal nuclear episomes. The proteins encoded by the E1 and E2 open reading frames (ORFs) load the cellular replication machinery at the origin of the HPV replication (reviewed in reference 6). The E1 protein is an origin recognition factor, which is loaded in a sequence-specific manner by the E2 protein at the replication origin, where it forms the E1 double-hexameric replicative helicase upon oligomerization (7-10). Before the initiation of replication, the oligomeric E1 protein unwinds dsDNA into two single strands, assembles into the double-hexameric, ATP-dependent replicative helicase, and loads the cellular replication complex at replication forks for the initiation of DNA replication (r...

Section: Fig 3 (A) U2os Cells Were Transfected With 250 Ng Of Hpv18 Esupporting

confidence: 80%

Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification

Reinson

Toots

Kadaja

et al. 2013

125

194

“…An alternative suggestion would be that HPV DNA maintenance replication in W12 is indeed E1-driven but that, in these cells, E1 is only able to license HPV DNA for replication once per S phase. Although there is no direct evidence to support the idea that E1's activity is limited to only once per S phase, the report by Deng et al (7) demonstrating that E1's localization to the nucleus is regulated by the cyclin E-cdk2 phosphorylation opens an avenue to this hypothesis. It is possible that the cell-cycle-regulated entry of E1 into the nucleus may confer once-per-S-phase activity to E1.…”

Section: Discussionmentioning

confidence: 72%

Different Modes of Human Papillomavirus DNA Replication during Maintenance

Hoffmann¹,

Hirt

Bechtold³

et al. 2006

Human papillomavirus (HPV) begins its life cycle by infecting the basal cells of the epithelium. Within these proliferating cells, the viral genomes are replicated, maintained, and passed on to the daughter cells. Using HPV episome-containing cell lines that were derived from naturally infected cervical tissues, we investigated the mode by which the viral DNAs replicate in these cells. We observed that, whereas HPV16 DNA replicated in an ordered once-per-S-phase manner in W12 cells, HPV31 DNA replicated via a random-choice mechanism in CIN612 cells. However, when HPV16 and HPV31 DNAs were separately introduced into an alternate keratinocyte cell line NIKS, they both replicated randomly. This indicates that HPV DNA is inherently capable of replicating by either random-choice or once-per-S-phase mechanisms and that the mode of HPV DNA replication is dependent on the cells that harbor the viral episome. High expression of the viral replication protein E1 in W12 cells converted HPV16 DNA replication to random-choice replication and, as such, it appears that the mode of HPV DNA replication in proliferating cells is dependent on the presence or the increased level of this protein in the host cell. The implications of these observations on maintenance, latency, and persistence are discussed.The host tissue of human papillomaviruses is the stratified epithelium. This tissue is complex in that it is composed of layered sheets of nondividing cells in various stages of terminal differentiation, with the uppermost layer being the most differentiated. Only cells of the bottom-most layer of this tissue, the basal cells, proliferate. Although the HPV life cycle begins with the infection of a basal cell, it only comes to completion when the infected cell reaches the upper layers of the epithelium. As a consequence, the HPV DNA initially finds itself in the nucleus of a proliferating cell, but later in that of a differentiating (nonproliferating) one. This sequential mixture of cell states that the virus has to contend with has undoubtedly shaped the way by which HPV replicates its DNA throughout the varying milieux during its life cycle.It is proposed that immediately after infection, the papillomavirus DNA copy number is amplified to a certain level (50 to 400) per cell (9). This first amplification replication is believed to be rapid and transient, after which the viral DNA is stably maintained at this level in subsequent divisions of the basal cell. This is thought to be achieved by maintenance replication, where the viral episomes approximately double their copy number during the S phase of the host cell cycle and segregate to the resulting two daughter cells. After differentiation of the host cell, the viral DNA undergoes another amplification step, the second amplification replication, which increases the HPV DNA copy number to several hundreds or thousands per cell. This is followed by packaging of the viral DNA into virus particles. Although this triphasic model has not been proven in its entirety, it is supported by va...

“…The T468A mutation does not affect the nucleocytoplasmic localization of the E1 protein, whereas E1 S89A, S93A, or S89,93A mutations, even in combination with an NES mutation, have much-reduced nuclear import relative to that of wild type E1, suggesting the initial need for cytoplasmic kinases for efficient nuclear import (27). The stretch of 50 amino acids (residues 81 to 130) has been designated the localization regulatory region (LRR); it contains the consensus cyclin binding motif, the three serine substrates for kinases specific for S/T-P, and the NES.…”

mentioning

confidence: 99%

“…and T468, each followed by a proline residue. S107 is located within a potent CRM1-dependent nuclear export se-quence (NES) (residues 96 to 115), and phosphorylation of this residue by cyclin E/cdk2 or cyclin A/cdk2 in vivo inactivates the NES (27). When cdk2 is inhibited by p21cip1, E1 is shuttled out of the nucleus.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mitogen-Activated Protein Kinases Activate the Nuclear Localization Sequence of Human Papillomavirus Type 11 E1 DNA Helicase To Promote Efficient Nuclear Import

Lin

Deng

et al. 2007

Self Cite

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...