Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

Gagnon, David; Sénéchal, Hélène; D’Abramo, Claudia M.; Álvarez, Jennifer; McBride, Alison A.; Archambault, Jacques

doi:10.1016/j.virol.2013.02.012

Cited by 9 publications

(14 citation statements)

References 43 publications

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“…Unlike the results obtained with HPV11, replacement of the conserved F594 or C596 with alanine had a dramatic effect on BPV1 DNA replication, decreasing replication levels by more than 90% (P Ͻ 0.0001) (Fig. 8B), consistent with what was reported for these substitutions in vitro (25). Unfortunately, we have been unable to assess the expression levels of any BPV1 E1 proteins (wild type or mutant) in these experiments due to the lack of a suitable antibody sensitive enough for immunoblotting.…”

Section: Figsupporting

confidence: 88%

“…8A) and analyzed the activities of the resulting mutant proteins in our previously described cell-based BPV1 DNA replication assay (which is based on the same luciferase readout used in the HPV11 assay shown in Fig. 2) (25). Unlike the results obtained with HPV11, replacement of the conserved F594 or C596 with alanine had a dramatic effect on BPV1 DNA replication, decreasing replication levels by more than 90% (P Ͻ 0.0001) (Fig.…”

Section: Figmentioning

confidence: 87%

“…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). In all experiments, the total amount of transfected DNA was adjusted to 100 ng with pCI (Promega) as the carrier DNA.…”

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%

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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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Section: Figsupporting

confidence: 88%

Section: Figmentioning

confidence: 87%

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%

See 2 more Smart Citations

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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“…This method, developed by the Archambault laboratory for both BPV-1 and HPV-31 in C33a cells, requires the cotransfection of plasmids encoding E2, the homologous E1 helicase, Renilla luciferase (internal control), and firefly luciferase (target replicon) (41,42). The firefly luciferase gene is constitutively expressed due to the presence of a cytomegalovirus (CMV) promoter.…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

Culleton

Kanginakudru

DeSmet

et al. 2017

J Virol

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Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-toglutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.IMPORTANCE Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

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Papillomavirus Replication

Culleton¹,

Androphy²,

Kanginakudru³

2015

Human Papillomavirus (HPV)-Associated Oropharyngeal Cancer

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Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1

Cited by 9 publications

References 43 publications

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

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

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions

Papillomavirus Replication

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