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1.
PLoS Pathog ; 9(7): e1003440, 2013.
Article in English | MEDLINE | ID: mdl-23853584

ABSTRACT

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.


Subject(s)
Adenosine Deaminase/metabolism , Host-Pathogen Interactions , Influenza A virus/physiology , Viral Nonstructural Proteins/metabolism , Virulence Factors/metabolism , Virus Replication , Adenosine Deaminase/chemistry , Adenosine Deaminase/genetics , Biological Transport , Cell Line , Dengue Virus/enzymology , Humans , Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/metabolism , Influenza, Human/pathology , Influenza, Human/virology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/pathology , Respiratory Mucosa/virology , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Species Specificity , Two-Hybrid System Techniques , Viral Nonstructural Proteins/genetics , Virulence Factors/genetics
2.
PLoS Pathog ; 8(6): e1002761, 2012.
Article in English | MEDLINE | ID: mdl-22761572

ABSTRACT

Human Papillomaviruses (HPV) cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV). To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes.


Subject(s)
Host-Parasite Interactions/genetics , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomavirus Infections/genetics , Cell Line , Genotype , Humans , Papillomaviridae/pathogenicity , Papillomavirus Infections/virology , Two-Hybrid System Techniques
3.
Mol Metab ; 76: 101772, 2023 10.
Article in English | MEDLINE | ID: mdl-37442376

ABSTRACT

OBJECTIVES: Readily accessible human pancreatic beta cells that are functionally close to primary adult beta cells are a crucial model to better understand human beta cell physiology and develop new treatments for diabetes. We here report the characterization of EndoC-ßH5 cells, the latest in the EndoC-ßH cell family. METHODS: EndoC-ßH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-ßH5 cells. We performed transcriptome, immunological and extensive functional assays. RESULTS: Ready to use EndoC-ßH5 cells display highly efficient glucose dependent insulin secretion. A robust 10-fold insulin secretion index was observed and reproduced in four independent laboratories across Europe. EndoC-ßH5 cells secrete insulin in a dynamic manner in response to glucose and secretion is further potentiated by GIP and GLP-1 analogs. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors. Cytokines induce a gene expression signature of inflammatory pathways and antigen processing and presentation. Finally, modified HLA-A2 expressing EndoC-ßH5 cells elicit specific A2-alloreactive CD8 T cell activation. CONCLUSIONS: EndoC-ßH5 cells represent a unique storable and ready to use human pancreatic beta cell model with highly robust and reproducible features. Such cells are thus relevant for the study of beta cell function, screening and validation of new drugs, and development of disease models.


Subject(s)
Insulin-Secreting Cells , Humans , Insulin-Secreting Cells/metabolism , Insulin Secretion , Cell Line , Insulin/metabolism , Transcription Factors/metabolism , Glucose/metabolism
4.
J Virol ; 85(24): 13010-8, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21994455

ABSTRACT

The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1, and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral genome. To identify these cellular partners, we generated a comprehensive physical interaction map between each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234 human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is required for the cellular antiviral response.


Subject(s)
Host-Pathogen Interactions , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza A Virus, H5N1 Subtype/pathogenicity , Protein Interaction Mapping , RNA-Dependent RNA Polymerase/metabolism , Viral Proteins/metabolism , Humans , Protein Binding , Two-Hybrid System Techniques , Virus Replication
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