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A preprint version has been deposited to Research Square at https://doi.org/10.21203/rs.3.rs-1430558/v1; International audience; Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.

International audience; The signaling pathways and cellular functions regulated by the four Numb-associated kinases (NAKs) are largely unknown. We previously reported that AAK1 and GAK control intracellular trafficking of RNA viruses, and recently revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE in this process have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell-based protein-protein interaction assays. We found that CLINT1, a cargo-specific adaptor implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 (T294) CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. In addition, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE's role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied NAK enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design.

Background: The activation of SRC kinases by trans-phosphorylation raises the question of their interplay. This work addresses this question by studying the interactions of SRC kinases with the SRC Kinase Adaptor Phosphoprotein 2 (SKAP2) by combining a luciferase complementation assay and extensive site-directed mutagenesis. Results: SKAP2 interacts with SRC kinases through two different module organizations that have different affinities for the activated and non-activated kinase conformations. The DIM domain of SKAP2 is necessary and sufficient to bind to most activated SRC kinases. A second SKAP2 module organization for binding to SRC kinases at their steady state is composed of the DIM and SH3 domains as well as the linker located between the PH and the SH3 domains. Classical binding sites of the PH and SH3 domains and the tyrosines located inside linkers modulate these interactions. Analysis of HCK mutants supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. Conclusion: In this article, we show that SKAP2 have a modular architecture composed of 2 domains and one interdomain to interact with SRC kinases, which binding capacity of each module depends on their localization and activation. This work opens new perspectives on how activation of SRC kinases occurs, which could have new and interesting therapeutic consequences.

Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.

Posté sur BioRxiv le 4 septembre 2020 https://www.biorxiv.org/content/10.1101/2020.09.03.282103v1.article-info; Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

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.

The world is facing a major health crisis, the global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus, for which no approved antiviral agents or vaccines are currently available. 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 via Addgene. 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.

The world is facing a major health crisis, the global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus, for which no approved antiviral agents or vaccines are currently available. 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 via Addgene. 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.

International audience; 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.

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

© The Author(s) 2019., 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., This work was supported by a Claudia Adams Barr Award to S.G.C., a Fonds de la Recherche Scientifique (F.R.S.- FNRS)-Télévie Grant and a Wallonia-Brussels International (WBI)-World Excellence Fellowship to J.O., NHGRI grants U41HG001715 awarded to M.V., D.E.H. and M.A.C, and P50HG004233 awarded to M.V. We acknowledge the support of Labex IBEID (10- LABX-0062). M.V. is a Chercheur Qualifié Honoraire from the Fonds de la Recherche Scientifique (FRS-FNRS, Wallonia-Brussels Federation, Belgium).

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 the concept of an “assayome” for binary protein-protein interaction (PPI) mapping as an optimal combination of assays and/or assay versions that maximizes detection of true positive interactions, while avoiding detection of random protein pairs. We engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions differing by protein expression systems and tagging configurations. The resulting N2H assayome 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 assayome concept should be applicable to systematic mapping of other biological landscapes.

// Jean-Francois Bureau 1, 2 , Patricia Cassonnet 3, 4, 5 , Laura Grange 1, 2 , Julien Dessapt 1, 2 , Louis Jones 3, 4, 5 , Caroline Demeret 3, 4, 5 , Anavaj Sakuntabhai 1, 2 and Yves Jacob 3, 4, 5 1 Unite de Genetique Fonctionnelle des Maladies Infectieuses, Departement Genome et Genetique, Institut Pasteur, Paris, France 2 CNRS URA3012, Paris, France 3 Unite de Genetique Moleculaire des Virus a ARN, Departement Virologie, Institut Pasteur, Paris, France 4 UMR3569, Centre National de la Recherche Scientifique, Paris, France 5 Universite Paris Diderot, Paris, France Correspondence to: Jean-Francois Bureau, email: jfb@pasteur.fr Yves Jacob, email: yves.jacob@pasteur.fr Keywords: luciferase complementation assay; protein-protein interaction; SRC-kinase family; adaptor; FYB; Pathology Received: November 16, 2017 Accepted: January 30, 2018 Published: February 06, 2018 ABSTRACT 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.

International audience; IntroductionLe Polyomavirus à cellules de Merkel (MCPyV) est reconnu depuis 2012 par l’IARC comme l’agent étiologique du carcinome à cellules de Merkel chez l’Homme. Cependant, les mécanismes conduisant à l’infection puis à l’oncogenèse viro-induite sont peu connus. Nos travaux ont pour objectifs d’identifier les partenaires cellulaires des oncoprotéines (antigènes T et t) du MCPyV afin de caractériser son virhostome (virus-host interactome).Matériel et méthodesL’identification des interactions entre les partenaires cellulaires et les protéines virales est réalisée par l’utilisation de la technologie du double hybride en levures puis validée à l’aide d’un test de complémentation en cellules de mammifère (HT-GPCA, High-Throughput Gaussia Princeps Complementation Assay).RésultatsGrâce à ces deux techniques, nous avons mis en évidence diverses interactions entre l’oncogène sT (small T) et des protéines cellulaires impliquées notamment dans le cycle cellulaire ou encore de la voie NFκB. Par ailleurs, nous avons identifié une interaction entre cet antigène et la protéine phosphatase PP2A, connue pour son implication dans des voies de signalisation oncogéniques, ainsi qu’avec l’oncogène LT (Large T). L’effet biologique de ces interactions sur les voies de signalisation est en cours de caractérisation.DiscussionCes résultats pourront nous permettre de comprendre la biologie du virus et les étapes de l’oncogenèse viro-induite afin d’identifier de potentielles cibles thérapeutiques.

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 aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. 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 exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes.

International audience

Objective: To evaluate the efficiency of human papillomavirus (HPV) testing by Hybrid Capture II (Digene Diagnostics Inc., Silver Spring, MD) with regard to detecting biopsy-confirmed cervical intraepithelial neoplasia (CIN) or high-grade CIN in women with mild atypia, compared with the efficiencies of polymerase chain reaction (PCR), Southern blot hybridization, and cytology. Methods: We prospectively studied 378 women with atypical squamous cells of undetermined significance (ASCUS) (n = 111) or low-grade squamous intraepithelial lesions (SILs) (n = 267) demonstrated by referral cytology. We did repeat cytology, sampling for detection of HPV DNA by Hybrid Capture II, PCR, and Southern blot hybridization, and colposcopic evaluation with cervical biopsies. Results: All participants underwent the Hybrid Capture II test and 320 underwent the three HPV tests. Sensitivities of Hybrid Capture II for detecting CIN and high-grade CIN (0.81 and 0.86, respectively) were similar to those of cytology (0.83 and 0.82, respectively) and PCR (0.77 and 0.95, respectively), and higher than those of Southern blot hybridization (0.48 and 0.45, respectively). Compared with cytology, combined triage with Hybrid Capture II improved sensitivities for detecting CIN (0.94 versus 0.83, P < .001) and high-grade CIN (0.96 versus 0.85), though the latter difference was not significant (P = .17). In women with ASCUS, sensitivities of combined triage and cytology for detecting CIN were 0.94 and 0.71, respectively (P = .01), and sensitivities of the two methods for detecting high-grade CIN were 0.92 and 0.66, respectively (P = .13). The increase in sensitivity was lower among women with low-grade SILs; for these women, cytology had high sensitivity (0.86 for CIN and 1.00 for high-grade CIN). The specificity of combined triage was significantly lower than that of cytology in both groups. Conclusion: Compared with repeat cytology, combined triage with HPV testing markedly improves sensitivity for detecting CIN in women with ASCUS, but at the expense of specificity.

International audience; We previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses.

International audience; 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.

International audience; 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.

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.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

SUMMARY 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.

International audience; 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.

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.

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.

International audience; 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%) β-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 β-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.

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 β1 (TGF-β1) 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-β1 signaling pathway could be a determinant in HPV5 skin carcinogenesis.

Les proprietes oncogeniques des HPV sont associees a l’expression de deux proteines virales E6 et E7. Ces proteines virales ne possedent pas d’activite enzymatique mais interagissent avec de nombreuses proteines cellulaires pour modifier leur activite, leur localisation, ou leur demi-vie. Les deux activites les plus decrites pour E6 et E7 sont la degradation de p53 et de pRB1 respectivement. Pour obtenir une carte plus complete des reseaux d’interactions proteiques, nous avons utilise une nouvelle approche d’interactomique, la PCA (protein fragment complementation assay). Celle ci est basee sur la reconstitution de l’activite de la Gaussia princeps luciferase a partir de deux fragments inactifs exprimes dans des cellules de mammiferes. Une banque de 650 ADNc codant pour le systeme ubiquitine-proteasome a ete criblee avec E6 et E7 par l’approche PCA. Nous avons identifie de nouveaux partenaires de E6 et E7 qui sont impliques dans la reponse immune innee et dans le controle de la proliferation cellulaire. L’impact de ces nouvelles interactions dans la biologie et la pathogenicite des HPV sera discute.

Part of this information was presented as a poster at the Second Wolrd Congress of Veterinary Dermatology in Montréal (Canada), 13-16 May 1992.; International audience; Cutaneous papillomavirus infection was diagnosed in a 6-year-old female Boxer dog that was under long-term corticosteroid therapy for atopic dermatitis. Multiple black, rounded papules were present on the ventral skin. Spontaneous regression occurred within 3 weeks after cessation of corticosteroids. Histologically, the lesions consisted of well-demarcated cup-shaped foci of epidermal endophytic hyperplasia with marked parakeratosis. In the upper stratum spinosum and in the stratum granulosum, solitary or small collections of enlarged keratinocytes were observed with basophilic intranuclear inclusion bodies and a single eosinophilic fibrillar cytoplasmic inclusion. Ultrastructurally, viruslike particles (40-45 nm in diameter) were observed within the nucleus, free or aggregated in crystalline arrays. Undulating fibrillar material, thought to be a modified keratin protein, was observed in the cytoplasmic inclusion. Immunohistochemistry, restriction enzyme analysis, and molecular hybridization experiments indicated that these distinctive clinical, histologic, and cytologic features were associated with a novel canine papillomavirus.

International audience; Patients suffering from epidermodysplasia verruciformis are prone to nonmelanoma skin cancers, due to an inherited abnormal susceptibility to the oncogenic human papillomavirus type 5. Genotoxic sunlight ultraviolet B radiations are likely to be a cofactor. Lesions of two human-papillomavirus-type-5-infected epidermodysplasia verruciformis patients collected during an 8 y period were retrospectively studied for p53 mutations in exons 5 through 8 by a polymerase chain reaction single-strand conformation polymorphism technique and/or by DNA sequencing of amplified exons. Mutations were detected in 11 of 26 (42.3%) specimens, including five (62.5%) squamous cell carcinomas, three (33.3%) Bowen's carcinomas in situ, two (40%) actinic keratoses, and one (33%) benign lesion. The nine mutations characterized by sequencing were shown to be missense and to affect mutational hotspots in human cancers. Five were C-->T transitions at dicytidine sites considered as ultraviolet signature mutations. Two were transversions (C-->G and C-->A) at dicytidine sites and two were C-->T transitions at nondipyrimidine sites. A marked p53 immunoreactivity was disclosed in 72.7% of 11 invasive carcinomas, 55.6% of nine carcinomas in situ, 37.5% of eight actinic keratoses, and one of three benign lesions. This includes 81.8% of 11 specimens with a p53 mutation but also 50% of 14 specimens with no mutation detected. A dysfunction of the p53 gene is thus likely to play a part in epidermodysplasia verruciformis carcinogenesis, either due to ultraviolet-B-induced p53 mutations, as in nonmelanoma skin cancers in the general population, or involving other mutagens or mechanisms. The part played by human papillomavirus type 5 proteins expressed in epidermodysplasia verruciformis keratinocytes remains to be determined.