mRNA Splicing Regulates Human Papillomavirus Type 11 E1 Protein Production and DNA Replication

Deng, Wentao; Jin, Ge; Lin, Biing-Yuan; Tine, Brian A. Van; Broker, Thomas R.; Chow, Louise T.

doi:10.1128/jvi.77.19.10213-10226.2003

Cited by 25 publications

(37 citation statements)

References 80 publications

(68 reference statements)

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“…We have shown that a fusion protein of GFP and HPV-11 E1 (GFP-H11 E1) supports transient replication as efficiently as the wild-type E1 protein (26). However, the majority of the transcripts from a native E1 or GFP-E1 fusion expression vector contain an intragenic splice from the preferred splice donor site at nt 847 (within the sixth codon) to nt 2622 near the carboxyl terminus, just upstream of the E2 open reading frame, which overlaps the E1 coding region.…”

Section: Resultsmentioning

confidence: 99%

“…Five micrograms of pMT2-H11 E2, 1 g of pGFP-H11 E1dm (wild type or mutation), and 0.5 g of HPV-11 ori-containing VOL. 81,2007 MAP KINASES ACTIVATE THE NLS OF HPV E1 DNA HELICASE 5067 plasmid were cotransfected into 293 cells by electroporation as described previously (26). The low-molecular-weight DNA was harvested 48 h posttransfection, using methods reported previously (14).…”

Section: Methodsmentioning

confidence: 99%

“…pmRFP-H11 E2 and pmRFP-H11 E2 K239A were made by swapping the enhanced green fluorescent protein (GFP) of the corresponding pGFP-H11 E2 clones (67,71) with the monomeric red fluorescent protein (mRFP) (9) between the Eco47III and BglII sites. The wild-type HPV-11 E1 construct contains GFP fused to its amino terminus and has mutations that disable the RNA splicing donor site at nucleotide (nt) 847 (GFP-H11 E1dm) without affecting the encoded E1 amino acid sequence (26). All of the E1 mutations were prepared by site-directed mutagenesis using PCR, built on this E1dm background, and cloned into the pGFP-C1 vector (Clontech, Mountain View, CA) between BamHI and KpnI restriction enzyme cleavage sites.…”

Section: Methodsmentioning

confidence: 99%

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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

J Virol

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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

J Virol

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“…Four cyclin kinase phosphorylation sites are present in E1 proteins, and mutation of these sites drastically reduces the replication activity of E1 (98). It is still not clear how E1 activity is regulated in differentiating cells, but studies with HPV-31 have shown that expression of E1 transcripts shifts from the early to late promoters, resulting in increased E1 expression (29). A shift in the absolute levels of E1 or the ratios of E1 to E2 is thought to result in high-level replication upon differentiation.…”

Section: E1 and E2 Replication Proteinsmentioning

confidence: 99%

Pathogenesis of Human Papillomaviruses in Differentiating Epithelia

Longworth

Laimins

2004

Microbiol Mol Biol Rev

551

545

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HUMAN PAPILLOMAVIRUSES AND CERVICAL CANCERHuman papillomaviruses (HPVs) are small, double-stranded DNA viruses that infect cutaneous and mucosal epithelial tissues of the anogenital tract, the hands, or the feet. A subset of HPV types are the causative agents of cervical cancer, since 99% of tumors are positive for HPV DNA (150). To date, over 100 different viral types have been identified, and about onethird of these infect epithelial cells in the genital tract. The viral types that infect the genital tract fall into two categories: high risk and low risk. The high-risk types are associated with the development of anogenital cancers including those of the cervix, while infections by the low-risk HPVs induce only benign genital warts. The high-risk types include HPV-16, HPV-18, HPV-31, HPV-33, and HPV-45, while the low-risk types are HPV-6 and HPV-11. HPVs that infect the genital tract are sexually transmitted, and it is estimated that about two-thirds of individuals who have sexual relations with an infected partner will themselves become infected. However, the majority of infections are subclinical (137). Infection by high-risk HPVs is not limited to the genital tract, since approximately 20% of cancers of the oropharynx contain DNA from these HPV types (61).Infection of the genital tract by HPVs can initially result in low-grade lesions termed dysplasias or cervical intraepithelial neoplasia grade I. These lesions exhibit only mildly altered patterns of differentiation, and many of them are cleared by the immune system in less than a year (62, 71). The mechanisms by which the cellular immune response clears HPV infections are still not clearly understood. Some of these lesions, however, are not cleared by the immune system and can persist for periods as long as several decades. Persistence of infection by high-risk HPV types is the greatest risk factor for development of genital malignancies such as squamous cell carcinoma or, less commonly, adenocarcinoma of the cervix (161). Cervical cancer is the second most prevalent cancer worldwide and is the fifth leading cause of cancer deaths in women (120,124). Approximately 470,000 new cases of cervical cancer are diagnosed yearly, with the mean age for the development of malignancy being 52 years (8, 124). Risk factors for tumor development include persistent infection with high-risk viral types, a large number of lifetime sexual partners, coinfection with human immunodeficiency virus, immunosuppression, and cigarette smoking (81). Most cases of cervical cancer are found outside of the United States and Western Europe. In the United States, the number of cases of cervical cancer has declined by over 80% in the last 50 years due to the implementation of the Pap smear as a diagnostic (137). While the number of cases has significantly decreased, approximately 10,000 women are diagnosed with cervical cancer and 5,000 die of this disease annually (120). HUMAN PAPILLOMAVIRUS LIFE CYCLEHPVs are nonenveloped viruses with icosahedral capsids that replicate their genomes wi...

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“…Plasmid p11E1, which expresses HPV11 E1 fused at its N terminus to green fluorescent protein (GFP), was constructed by inserting an XhoI-BamHI fragment including the E1 open reading frame (ORF) between the XhoI and BamHI sites of pEGFP-C2 (Clontech). The XhoI-BamHI E1 fragment used for this construction contains the previously reported mutations inactivating the major splicing donor site (21), as well as a NotI restriction site between the stop codon of E1 and the BamHI site. The nucleotide sequence of this fragment can be summarized as follows, with the XhoI, NotI, and BamHI sites underlined and the translation initiation and termination codons of the E1 ORF in brackets and italicized: 5=-CTCGAGCCACC[ATG-E1ORF-TAG]TGAGCGGCCGCTAGTAACATATGTAGTAAGGATCC-3=.…”

Section: Methodsmentioning

confidence: 99%

Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

Bergvall

Gagnon

Titolo

et al. 2016

J Virol

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The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCEWhile much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural integrity of the helicase domain, followed by an acidic region (AR) and a C-terminal tail (C-tail) that have been shown to regulate the oligomerization of BPV1 E1 in vitro. Characterization of E1 chimeras revealed that, while the function of the AR could be transferred from BPV1 E1 to HPV11 E1, that of the C-tail could not. These results suggest that the E1 CTD performs multiple functions in DNA replication, some of them in a virus type-specific manner.H uman papillomaviruses (HPVs) are small viruses with double-stranded DNA (dsDNA) genomes that infect both outer and mucosal stratified epithelium (for a recent review see reference 1). Of the more than 180 HPV genotypes described to date, about 20% infect the anogenital tract, causing either benign or precancerous lesions (2). In addition to their well-established role in inducing cervical and other anogenital cancers, HPVs have more recently been shown to be the causative agents for the majority of oropharyngeal cancers (3, 4).Only two HPV proteins, E1 and E2, are required for replicati...

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mRNA Splicing Regulates Human Papillomavirus Type 11 E1 Protein Production and DNA Replication

Cited by 25 publications

References 80 publications

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

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

Pathogenesis of Human Papillomaviruses in Differentiating Epithelia

Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

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