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1.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
2.
Nat Commun ; 10(1): 3907, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467278

RESUMO

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.


Assuntos
Bioensaio/métodos , Mapeamento de Interação de Proteínas/métodos , Proteoma/análise , Bases de Dados de Proteínas , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Mapas de Interação de Proteínas , Proteínas/metabolismo , Proteômica/métodos , Técnicas do Sistema de Duplo-Híbrido
3.
Oncotarget ; 9(17): 13102-13115, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568343

RESUMO

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) is a broadly expressed adaptor associated with the control of actin-polymerization, cell migration, and oncogenesis. After activation of different receptors at the cell surface, this dimeric protein serves as a platform for assembling other adaptors such as FYB and some SRC family kinase members, although these mechanisms are still poorly understood. The goal of this study is to map the SKAP2 interactome and characterize which domains or binding motifs are involved in these interactions. This is a prerequisite to finely analyze how these pathways are integrated in the cell machinery and to study their role in cancer and other human diseases when this network of interactions is perturbed. In this work, the domain and the binding motif of fourteen proteins interacting with SKAP2 were precisely defined and a new interactor, FAM102A was discovered. Herein, a fine-tuning between the binding of SRC kinases and their activation was identified. This last process, which depends on SKAP2 dimerization, indirectly affects the binding of FYB protein. Analysis of conformational changes associated with activation/inhibition of SRC family members, presently limited to their effect on kinase activity, is extended to their interactive network, which paves the way for therapeutic development.

4.
mSphere ; 2(6)2017.
Artigo em Inglês | MEDLINE | ID: mdl-29202037

RESUMO

The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus's adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.

5.
Sci Rep ; 7(1): 16129, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170442

RESUMO

PKR is a cellular kinase involved in the regulation of the integrative stress response (ISR) and pro-inflammatory pathways. Two N-terminal dsRNA Binding Domains (DRBD) are required for activation of PKR, by interaction with either dsRNA or PACT, another cellular DRBD-containing protein. A role for PKR and PACT in inflammatory processes linked to neurodegenerative diseases has been proposed and raised interest for pharmacological PKR inhibitors. However, the role of PKR in inflammation is subject to controversy. We identified the flavonoid luteolin as an inhibitor of the PKR/PACT interaction at the level of their DRBDs using high-throughput screening of chemical libraries by homogeneous time-resolved fluorescence. This was further validated using NanoLuc-Based Protein Complementation Assay. Luteolin inhibits PKR phosphorylation, the ISR and the induction of pro-inflammatory cytokines in human THP1 macrophages submitted to oxidative stress and toll-like receptor (TLR) agonist. Similarly, luteolin inhibits induction of pro-inflammatory cytokines in murine microglial macrophages. In contrast, luteolin increased activation of the inflammasome, in a PKR-independent manner. Collectively, these data delineate the importance of PKR in the inflammation process to the ISR and induction of pro-inflammatory cytokines. Pharmacological inhibitors of PKR should be used in combination with drugs targeting directly the inflammasome.


Assuntos
Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , eIF-2 Quinase/metabolismo , Células HEK293 , Humanos , Inflamação/imunologia , Fosforilação/genética , Fosforilação/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/genética , eIF-2 Quinase/genética
6.
FEBS J ; 284(19): 3171-3201, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28786561

RESUMO

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system.


Assuntos
Interações Hospedeiro-Patógeno , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina/genética , Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Biologia Computacional , Regulação da Expressão Gênica , Genes Reporter , Papillomavirus Humano 16/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Anotação de Sequência Molecular , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Biblioteca de Peptídeos , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Replicação Viral
7.
Nat Methods ; 12(8): 787-93, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26053890

RESUMO

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this end, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to 1,000 domain-motif equilibrium binding affinities per day. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from human papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human 'PDZome'. We obtained sharply sequence-dependent binding profiles that quantitatively describe the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has wide potential for quantifying the specificities of interactomes.


Assuntos
Ensaios de Triagem em Larga Escala , Domínios PDZ , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Motivos de Aminoácidos , Cromatografia , Proteínas de Ligação a DNA/química , Humanos , Cinética , Ligantes , Proteínas Oncogênicas Virais/química , Conformação Proteica , Proteoma , Proteínas Repressoras/química , Biologia de Sistemas
8.
J Vis Exp ; (77): e50404, 2013 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-23893119

RESUMO

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors. This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells. A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Técnicas do Sistema de Duplo-Híbrido , Arecaceae/enzimologia , Células HEK293 , Humanos , Luciferases/química , Luciferases/metabolismo , Transfecção/métodos , Leveduras/genética , Leveduras/metabolismo
9.
Methods ; 58(4): 349-59, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22898364

RESUMO

Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or "interologs". Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus-host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interaction assays: yeast two-hybrid (Y2H), and our recently described "high-throughput Gaussia princeps protein complementation assay" (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split G. princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus-host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism-host interactions.


Assuntos
Alphapapillomavirus/fisiologia , Interações Hospedeiro-Patógeno , Luciferases/genética , Proteínas de Plantas/genética , Técnicas do Sistema de Duplo-Híbrido , Alphapapillomavirus/genética , Arecaceae/enzimologia , Biomarcadores/metabolismo , Análise por Conglomerados , Genótipo , Células HEK293 , Humanos , Luciferases/biossíntese , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Filogenia , Proteínas de Plantas/biossíntese , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tropismo Viral
10.
J Virol ; 86(6): 3121-34, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22258240

RESUMO

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.


Assuntos
Infecções por Alphavirus/metabolismo , Infecções por Alphavirus/virologia , Vírus Chikungunya/metabolismo , Interações Hospedeiro-Patógeno , Mapas de Interação de Proteínas , Proteínas não Estruturais Virais/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Febre de Chikungunya , Vírus Chikungunya/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas não Estruturais Virais/genética
12.
Microbiol Mol Biol Rev ; 73(2): 348-70, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19487731

RESUMO

Infections by human papillomaviruses (HPVs) are the most frequently occurring sexually transmitted diseases. The crucial role of genital oncogenic HPV in cervical carcinoma development is now well established. In contrast, the role of cutaneous HPV in skin cancer development remains a matter of debate. Cutaneous beta-HPV strains show an amazing ubiquity. The fact that a few oncogenic genotypes cause cancers in patients suffering from epidermodysplasia verruciformis is in sharp contrast to the unapparent course of infection in the general population. Our recent investigations revealed that a natural barrier exists in humans, which protects them against infection with these papillomaviruses. A central role in the function of this HPV-specific barrier is played by a complex of the zinc-transporting proteins EVER1, EVER2, and ZnT-1, which maintain cellular zinc homeostasis. Apparently, the deregulation of the cellular zinc balance emerges as an important step in the life cycles not only of cutaneous but also of genital HPVs, although the latter viruses have developed a mechanism by which they can break the barrier and impose a zinc imbalance. Herein, we present a previously unpublished list of the cellular partners of EVER proteins, which points to future directions concerning investigations of the mechanisms of action of the EVER/ZnT-1 complex. We also present a general overview of the pathogenesis of HPV infections, taking into account the latest discoveries regarding the role of cellular zinc homeostasis in the HPV life cycle. We propose a potential model for the mechanism of function of the anti-HPV barrier.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Membrana/metabolismo , Papillomaviridae/patogenicidade , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Feminino , Predisposição Genética para Doença , Homeostase , Humanos , Papillomaviridae/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Zinco/metabolismo
13.
J Exp Med ; 205(1): 35-42, 2008 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-18158319

RESUMO

Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses.


Assuntos
Epidermodisplasia Verruciforme/metabolismo , Epidermodisplasia Verruciforme/virologia , Regulação da Expressão Gênica , Proteínas de Membrana/fisiologia , Papillomaviridae/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/prevenção & controle , Zinco/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Predisposição Genética para Doença , Genoma Viral , Humanos , RNA Interferente Pequeno/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Zinco/química
14.
J Gen Virol ; 88(Pt 7): 1928-1933, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17554024

RESUMO

We identified sequences from two distantly related papillomaviruses in genital warts from two Burmeister's porpoises, including a PV antigen-positive specimen, and characterized Phocoena spinipinnis papillomavirus type 1 (PsPV-1). The PsPV-1 genome comprises 7879 nt and presents unusual features. It lacks an E7, an E8 and a bona fide E5 open reading frame (ORF) and has a large E6 ORF. PsPV-1 L1 ORF showed the highest percentage of nucleotide identity (54-55 %) with human papillomavirus type 5, bovine papillomavirus type 3 (BPV-3) and Tursiops truncatus papillomavirus type 2 (TtPV-2). This warrants the classification of PsPV-1 as the prototype of the genus Omikronpapillomavirus. PsPV-1 clustered with TtPV-2 in the E6 and E1E2 phylogenetic trees and with TtPV-2 and BPV-3 in the L2L1 tree. This supports the hypothesis that PV evolution may not be monophyletic across all genes.


Assuntos
Condiloma Acuminado/veterinária , Papillomaviridae/genética , Papillomaviridae/isolamento & purificação , Phocoena/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Condiloma Acuminado/virologia , DNA Viral/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Papillomaviridae/classificação , Filogenia , Especificidade da Espécie , Proteínas Virais/genética
15.
Acta Ophthalmol Scand ; 85(5): 551-6, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17362365

RESUMO

PURPOSE: Epithelial tumours of the lacrimal sac are rare but important entities that may carry grave prognoses. In this study the prevalence and possible role of human papillomavirus (HPV) infection in epithelial tumours of the lacrimal sac were evaluated. METHODS: Five papillomas and six carcinomas of the lacrimal sac were investigated for the presence of HPV using the polymerase chain reaction (PCR) technique. Fifteen specimens of dacryocystitis were included in the PCR reactions as controls. Furthermore, DNA in situ hybridization (ISH) and RNA ISH were performed. RESULTS: Low-risk HPV types 6 or 11 were identified in all four lacrimal sac papillomas suitable for PCR analysis and in situ hybridization. Four of six lacrimal sac carcinomas harboured HPV. One carcinoma was positive for HPV 11 only, two carcinomas had concomitant infection with HPV 6 or 11 and high-risk HPV 16, and the remaining carcinoma was positive for HPV 16. All specimens of dacryocystitis were betaglobin-positive and HPV-negative. Using DNA ISH, two papillomas and a single carcinoma showed evidence for vegetative HPV 11 DNA replication, whereas no HPV 16 DNA replication was found in the five carcinomas tested. HPV 11 RNA was demonstrated in two papillomas. CONCLUSIONS: By analysing 11 epithelial lacrimal sac papillomas and carcinomas using PCR, DNA ISH and RNA ISH, we found HPV DNA in all investigated transitional epithelium tumours of the lacrimal sac. HPV RNA was present in two of eight epithelial lacrimal sac tumours positive for HPV DNA. As RNA degrades fast in paraffin-embedded tissue, only a small fraction of DNA-positive tumours can be expected to be RNA-positive. We therefore suggest that HPV infection is associated with the development of lacrimal sac papillomas and carcinomas.


Assuntos
Carcinoma/virologia , Neoplasias Oculares/virologia , Papillomavirus Humano 11/isolamento & purificação , Papillomavirus Humano 6/isolamento & purificação , Doenças do Aparelho Lacrimal/virologia , Papiloma/virologia , Infecções por Papillomavirus/virologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autorradiografia , Carcinoma de Células Escamosas/virologia , Carcinoma de Células de Transição/virologia , Replicação do DNA , DNA Viral/análise , Feminino , Papillomavirus Humano 11/genética , Papillomavirus Humano 6/genética , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/análise
16.
Br J Ophthalmol ; 91(8): 1014-5, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17166894

RESUMO

AIM: To examine conjunctival papilloma and normal conjunctival tissue for the presence of human papillomavirus (HPV). METHODS: Archival paraffin wax-embedded tissue from 165 conjunctival papillomas and from 20 histological normal conjunctival biopsy specimens was analysed for the presence of HPV by PCR. Specimens considered HPV positive using consensus primers, but with a negative or uncertain PCR result using type-specific HPV probes, were analysed with DNA sequencing. RESULTS: HPV was present in 86 of 106 (81%) beta-globin-positive papillomas. HPV type 6 was positive in 80 cases, HPV type 11 was identified in 5 cases and HPV type 45 was present in a single papilloma. All the 20 normal conjunctival biopsy specimens were beta-globin positive and HPV negative. CONCLUSION: There is a strong association between HPV and conjunctival papilloma. The study presents the largest material of conjunctival papilloma investigated for HPV and the first investigation of HPV in normal conjunctival tissue. HPV types 6 and 11 are the most common HPV types in conjunctival papilloma. This also is the first report of HPV type 45 in conjunctival papilloma.


Assuntos
Neoplasias da Túnica Conjuntiva/virologia , Papiloma/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/virologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Túnica Conjuntiva/patologia , Neoplasias da Túnica Conjuntiva/patologia , Humanos , Pessoa de Meia-Idade , Papiloma/patologia , Infecções por Papillomavirus/patologia , Reação em Cadeia da Polimerase/métodos
17.
J Virol ; 80(24): 12420-4, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17020941

RESUMO

Mechanisms of cellular transformation associated with human papillomavirus type 5 (HPV5), which is responsible for skin carcinomas in epidermodysplasia verruciformis (EV) patients, are poorly understood. Using a yeast two-hybrid screening and molecular and cellular biology experiments, we found that HPV5 oncoprotein E6 interacts with SMAD3, a key component in the transforming growth factor beta1 (TGF-beta1) signaling pathway. HPV5 E6 inhibits SMAD3 transactivation by destabilizing the SMAD3/SMAD4 complex and inducing the degradation of both proteins. Interestingly, the E6 protein of nononcogenic EV HPV9 failed to interact with SMAD3, suggesting that downregulation of the TGF-beta1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.


Assuntos
Alphapapillomavirus/metabolismo , Regulação para Baixo , Proteínas Oncogênicas Virais/metabolismo , Transdução de Sinais/fisiologia , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Alphapapillomavirus/genética , Humanos , Transdução de Sinais/genética , Técnicas do Sistema de Duplo-Híbrido
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