Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1.
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ABSTRACT:
SUBMITTER: Gagnon D
PROVIDER: S-EPMC4654618 | biostudies-literature | 2013 May
REPOSITORIES: biostudies-literature
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Project description:Recent studies have suggested that the tethering of viral genomes to host cell chromosomes could provide one of the ways to achieve their nuclear retention and partitioning during extrachromosomal maintenance in dividing cells. The data we present here provide firm evidence that the partitioning of the bovine papillomavirus type 1 (BPV1) genome is dependent on the chromatin attachment process mediated by viral E2 protein and its multiple binding sites. On the other hand, the attachment of E2 and the E2-mediated tethering of reporter plasmids to host chromosomes are not necessarily sufficient for efficient partitioning, suggesting that additional E2-dependent activities might be involved in the latter process. The activity of E2 protein in chromatin attachment and partitioning is more sensitive to the point mutations in the N-terminal domain than its transactivation and replication initiation functions. Therefore, at least part of the interactions of the E2 N-terminal domain with its targets during the chromatin attachment and partitioning processes are likely to involve specific receptors not involved in transactivation and replication activities of the protein. The mutational analysis also indicates that the binding of E2 to chromatin is not achieved through interaction of linear N-terminal subsequences of the E2 protein with putative receptors. Instead, the composite surface elements of the N-terminal domain build up the receptor-binding surface of E2. In this regard, the interaction of BPV1 E2 with its chromosomal targets clearly differs from the interactions of LANA1 protein from Kaposi's sarcoma-associated human herpesvirus and EBNA1 from Epstein-Barr virus with their specific receptors.
| S-EPMC369436 | biostudies-literature
Project description:The transactivation domain (AD) of bovine papillomavirus type 1 E2 stimulates gene expression and DNA replication. To identify cellular proteins that interact with this 215-amino-acid domain, we used a transactivation-defective mutant as bait in the yeast two-hybrid screen. In vitro and in vivo results demonstrate that the cDNA of one plasmid isolated in this screen encodes a 37-kDa nuclear protein that specifically binds to an 82-amino-acid segment within the E2 AD. Mutants with point mutations within this E2 domain were isolated based on their inability to interact with AMF-1 and were found to be unable to stimulate transcription. These mutants also exhibited defects in viral DNA replication yet retained binding to the viral E1 replication initiator protein. Overexpression of AMF-1 stimulated transactivation by both wild-type E2 and a LexA fusion to the E2 AD, indicating that AMF-1 is a positive effector of the AD of E2. We conclude that interaction with AMF-1 is necessary for the transcriptional activation function of the E2 AD in mammalian cells.
| S-EPMC232578 | biostudies-other
Project description:The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor.
| S-EPMC189983 | biostudies-other
Project description:The bovine papillomavirus type 1 (BPV-1) E2 protein is the master regulator of papillomavirus replication and transcription. We have raised a panel of monoclonal antibodies (MAbs) against the BPV-1 E2 protein and used them to probe the structure and function of the protein. Five MAbs reacted with linear epitopes, and four MAbs recognized conformation-dependent epitopes which mapped within the C-terminal DNA-binding and dimerization domain. MAb 1E2 was able to recognize the replication- and transactivation-defective but not the competent conformation of the transactivation domain of the E2 protein. MAb 5H4 prevented efficiently the formation of E2-DNA as well as E2-dependent E1-E2-origin complexes and also dissociated preformed complexes in a concentration-dependent manner. Cotransfection of several MAbs with the BPV-1 minimal origin plasmid pUCAlu into CHO4.15 cells resulted in a dose-dependent inhibition of replication. Inhibition of replication by MAb 5H4 and the Fab' fragment of 5H4 correlated with their ability to dissociate the E2 protein from the DNA. MAb 3F12 and MAbs 1H10 and 1E4, directed against the hinge region, were also capable of inhibiting BPV-1 origin replication in CHO4.15 cells. However, the Fab' fragments of 1H10 and 3F12 had no effect in the transient replication assay. These data suggest that MAbs directed against the hinge region sterically hinder the inter- or intramolecular interactions required for the replication activity of the E2 protein.
| S-EPMC112508 | biostudies-literature
Project description:Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors.
| S-EPMC3187478 | biostudies-literature
Project description:BackgroundXinjiang is one of the regions with a high incidence of cervical cancer, and the genetic variation of human papillomavirus may increase its ability to infect the human body and enhance virus-mediated immune escape ability.MethodsSanger sequencing of the HPV16 genome from 165 samples positive for HPV16 infection and phylogenetic analysis of the E1 and E2 genes revealed the gene polymorphism of HPV16 in Xinjiang.ResultsThe results showed that there were 109 samples with variations in HPV16 E1, 48 sites with nucleotide variations (19 missense variations and 29 synonymous variations), and 91 samples with variations in HPV16 E2, 25 sites with nucleotide variations (20 missense variations and five synonymous variations).ConclusionsFrom the phylogenetic tree results, 149 samples were of the European variant and 16 samples were of the Asian variant. No African or North American/Asian variant types were found.
| S-EPMC8590209 | biostudies-literature
Project description:The E2 proteins of several papillomaviruses link the viral genome to mitotic chromosomes to ensure retention and the efficient partitioning of genomes into daughter cells following cell division. Bovine papillomavirus type 1 E2 binds to chromosomes in a complex with Brd4, a cellular bromodomain protein. Interaction with Brd4 is also important for E2-mediated transcriptional regulation. The transactivation domain of E2 is crucial for interaction with the Brd4 protein; proteins lacking or mutated in this domain do not interact with Brd4. However, we found that the C-terminal DNA binding/dimerization domain of E2 is also required for efficient binding to Brd4. Mutations that eliminated the DNA binding function of E2 had no effect on the ability of E2 to interact with Brd4, but an E2 protein with a mutation that disrupted C-terminal dimerization bound Brd4 with greatly reduced efficiency. Furthermore, E2 proteins in which the C-terminal domains were replaced with the dimeric DNA binding domain of EBNA-1 or Gal4 bound efficiently to the Brd4 protein, but the replacement of the E2 C-terminal domain with a monomeric red fluorescent protein did not rescue efficient Brd4 binding. Thus, E2 bound to Brd4 most efficiently as a dimer. To prove this finding further, the E2 DNA binding domain was replaced with an FKBP12-derived domain in which dimerization was regulated by a bivalent ligand. This fusion protein bound Brd4 efficiently only in the presence of the ligand, confirming that a dimer of E2 was required. Correspondingly, E2 proteins that could dimerize were able to bind to mitotic chromosomes much more efficiently than monomeric E2 polypeptides.
| S-EPMC2493320 | biostudies-literature
Project description:Elastic network models are used for investigation of the p53 core domain functional dynamics. Global modes of motion indicate high positive correlations for residue fluctuations across the A-B interface, which are not observed at the B-C interface. Major hinge formation is observed at the A-B interface upon dimerization indicating stability of the A-B dimer. These findings imply A-B as the native dimerization interface, whereas B-C is the crystal interface. The A-B dimer exhibits an opening-closing motion about DNA, supporting the previously suggested clamp-like model of nonspecific DNA binding followed by diffusion. Monomer A has limited positive correlations with DNA, while monomer B exhibits high positive correlations with DNA in the functionally significant slow modes. Thus, monomer B might seem to maintain the stability of the dimer-DNA complex by forming the relatively fixed arm of the dimer clamp, whereas the other arm of the clamp, monomer A, might allow sliding via continuous association/dissociation mechanisms.
| S-EPMC1483080 | biostudies-literature
Project description:Equine sarcoids are locally aggressive fibroblastic neoplasms considered to be the most common skin tumors of horses worldwide. Bovine papillomavirus types 1 and 2 have typically been associated with sarcoids in equids. Investigations aiming to identify papillomavirus strains, aside from bovine papillomaviruses 1 and 2, which might be associated with sarcoid lesions, have been lacking. The aim of this article is to report the identification of a third bovine papillomavirus type, bovine papillomavirus 13, associated with equine sarcoids. Six sarcoid lesions were collected from diverse anatomical sites on two horses from southern Brazil. To detect a broad spectrum of papillomavirus strains, eight degenerate primer pairs designed to detect conserved regions on the L1 and E1 genes were tested on the DNA samples. Direct sequencing was then performed on the obtained amplicons, and sequence identities were compared with sequences from all bovine papillomavirus types. The FAP59/FAP64, MY09/MY11, and AR-E1F2/AR-E1R4 sequences generated from the sarcoids were shown to present 99 to 100% identity with bovine papillomavirus 13, a new bovine papillomavirus type previously described in cattle. The results from this study suggest that there is a need to identify bovine papillomavirus type 13 and other papillomavirus strains that might be associated with sarcoids in diverse geographical areas; such investigations might establish the frequency of occurrence of this viral type in these common tumors of equids.
| S-EPMC3697707 | biostudies-literature
Project description: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-to-glutamate (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.
| S-EPMC5215344 | biostudies-literature