ViralORFeome: an integrated database to generate a versatile collection of viral ORFs

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® 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

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

BACKGROUND: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. RESULTS: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. CONCLUSIONS: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome

A novel genital human papillomavirus (HPV), HPV type 74, found in immunosuppressed patients.

The genome of a novel human papillomavirus (HPV) type, HPV74, was cloned from an iatrogenically immunosuppressed woman with persisting low-grade vaginal intraepithelial neoplasia. HPV74 was found to be phylogenetically related to the low-risk HPV types 6, 11, 44, and 55. HPV74 or a variant of this type was found in specimens from three additional immunosuppressed women but not in about 3,000 anogenital specimens from immunocompetent patients

<em>Virus-host interactome</em> des oncoprotéines et des protéines de capside du polyomavirus à cellules de Merkel

National audienc

Multiple Pigmented Cutaneous Papules Associated with a Novel Canine Papillomavirus in an Immunosuppressed Dog

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 audienceCutaneous 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

Genital warts in Burmeister's porpoises: characterization of <i>Phocoena spinipinnis</i> papillomavirus type 1 (PsPV-1) and evidence for a second, distantly related PsPV

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

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Higly Connected Viral Component

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 a 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) to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral non-structural protein nsP2, were identified and further validated by protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of literature for 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 involve heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shut-off as previously reported for other Old-World alphaviruses, and determined that among binding partners identified by yeast two-hybrid, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides a first interaction map between CHIKV and human proteins, and involves new host cell proteins in the replication cycle of this virus.info:eu-repo/semantics/publishe

Comparative Profiling of Ubiquitin Proteasome System Interplay with Influenza A Virus PB2 Polymerase Protein Recapitulating Virus Evolution in Humans

&lt;p&gt;The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions&lt;br&gt; (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&#039;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.&lt;br&gt; &lt;br&gt; &amp;nbsp;&lt;/p&gt;</p