A Quantitative and High-Throughput Assay of Human Papillomavirus DNA Replication

Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

doi:10.1007/978-1-4939-2013-6_23

Cited by 5 publications

(9 citation statements)

References 17 publications

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“…The HPV31 DNA replication assay was performed as described previously (25,27). Briefly, C33A cells were seeded at a density of 25,000 cells/well in white, flatbottom, 96-well plates and transfected 24 h later with a mix of four plasmids: an origin-containing plasmid with a firefly luciferase reporter in cis (pFLORI31), a Renilla luciferase plasmid as an internal control (pRL), and the indicated quantities of E1 and E2 expression vectors.…”

Section: Methodsmentioning

confidence: 99%

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

Gagnon

Lehoux

Archambault

2015

J Virol

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The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCEThe E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. H uman papillomaviruses (HPVs) are small DNA tumor viruses that infect squamous epithelia of the skin and mucosa. Infections by these viruses can lead to the development of benign and malignant tumors, depending on the viral types. Several HPV types are sexually transmitted and the causative agents of cervical cancer, the second most common cancer in women worldwide. Th...

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Section: Methodsmentioning

confidence: 99%

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

Gagnon

Lehoux

Archambault

2015

J Virol

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“…The HPV11 DNA replication assay was developed and performed essentially as described previously for HPV31 and BPV1 (22,24,25). Briefly, 2.5 ϫ 10 4 C33A cells/well were seeded in white flat-bottom 96-well plates (Corning) and transfected, 24 h later, with the following four plasmids: for HPV11, p11E1 (10 ng), p11E2 (10 ng), pFLORI11 (2.5 ng), and the Renilla luciferase (RLuc) control plasmid pRL (0.5 ng) (22); for BPV1, pCG E1Eag 1235 Ϫ (10 ng; BPV1 E1 expression plasmid [23]), pBPV1E2 (10 ng [25]), pFLORI-BPV1 short (2.5 ng [25]), and pRL (0.5 ng).…”

Section: Methodsmentioning

confidence: 99%

“…The LUMIER (luminescence-based mammalian interactome mapping) E1-E2 coimmunoprecipitation assay was adapted from Blasche and Koegl (26) using plasmid p11E1 and its derivatives, together with a previously described plasmid encoding HPV11 E2 fused to Renilla luciferase (RLuc-E2) (24). Briefly, 8.5 ϫ 10 5 C33A cells were transfected with 2 g of p11E1 expression plasmid, along with 2 g of RLuc-E2 expression plasmid, in a well of a 6-well plate.…”

Section: Methodsmentioning

confidence: 99%

“…2) (22,24,25). We chose the HPV11 system to evaluate the in vivo activity of the E1 CTD because it has proven to be more amenable to subsequent biochemical studies.…”

Section: Deletion Of the C Terminus Of E1 Is Detrimental To Hpv Dna Rmentioning

confidence: 99%

“…DNA replication levels are presented as FLuc/RLuc ratios, measured in C33A cells that were cotransfected with 2.5 ng of pFLORI11 and 0.5 ng of pRL, together with (ϩ) or without (Ϫ) 10 ng of p11E1 (E1) and 10 ng p11E2 (E2), as indicated. DNA replication levels were measured at different time points posttransfection (24,48, and 72 h). Standard deviations are indicated by the error bars.…”

Section: Deletion Of the C Terminus Of E1 Is Detrimental To Hpv Dna Rmentioning

confidence: 99%

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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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Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

Feng

Chen

et al. 2021

Virol. Sin.

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We have previously reported that bovine papillomavirus type 1 (BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae (budding yeast) cultures (Zhao and Frazer 2002, Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication (day 3–16), middle weak replication (day 23–34/45) and late stable replication (day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs (cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.

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A Quantitative and High-Throughput Assay of Human Papillomavirus DNA Replication

Cited by 5 publications

References 17 publications

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

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

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

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