pISTil: a pipeline for yeast two-hybrid Interaction Sequence Tags identification and analysis

Pellet, Johann; Meyniel, Laurène; Vidalain, Pierre‐Olivier; Chassey, Benoı̂t de; Tafforeau, Lionel; Lotteau, Vincent; Rabourdin–Combe, Chantal

doi:10.1186/1756-0500-2-220

Cited by 11 publications

(10 citation statements)

References 18 publications

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“…Positive clones were maintained onto this selective medium for 15 days to eliminate any contaminant AD-cDNA plasmid. AD-cDNAs were PCR-amplified, sequenced and analyzed using pISTil [69]. Cellular ORFs (interacting domains found in Y2H screens) were amplified from a pool of human cDNA libraries or from a plasmid encoding the corresponding cDNA from the MGC collection (IMAGE consortium) using KOD polymerase (Toyobo) and cloned by recombinational cloning into pDONR207 (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

et al. 2013

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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.

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Section: Methodsmentioning

confidence: 99%

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

et al. 2013

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“…To establish a standardized system to analyze these ISTs, we developed pISTil, a bioinformatics pipeline combined to a user-friendly Web interface (see Note 30) (18). The pISTil system is highly flexible and allows (a) systematic and fast assignation of ISTs to a unique protein accession number; (b) annotation of "in-frame" or not-in-frame ISTs; (c) manual checking and visualization of annotated ISTs through a userfriendly Web interface; and (d) export of ppi in multiple formats, such as MIMIx standard format (19).…”

Section: Grow Each Yeast Bait Colony Overnight In 2 ML Sd-w Under Agimentioning

confidence: 99%

Virus–Human Cell Interactomes

Tafforeau¹,

Rabourdin–Combe²,

Lotteau³

2011

Methods in Molecular Biology

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Using global approaches and high-throughput technologies in virology brings a new vision of the infections physiology and allows the identification of cellular factors, mandatory for viral life cycle, that could be targeted by original therapeutic agents. It opens perspectives for the treatment of viral infections by acting on cellular pathways that the virus must use for its own replication. Combining these new molecules with classical antiviral drugs and immunomodulators diversifies and enlarges the antiviral arsenal and contributes to fight drug resistance.Our laboratory and others are constructing virus-human interactomes to propose a comprehensive analysis of viral infection at the cellular level. Studying these infection maps, where the viral infection can be visualized as perturbation of the human protein-protein interaction network, and identifying the biological functions that are impaired by these perturbations may lead to discovery of new therapeutic targets. These virus-human interaction maps are constructed in a stringent yeast two-hybrid system by screening human cDNA libraries with viral proteins as bait and integrating interactions mined from literature and public databases. Key words:Yeast two-hybrid screen, Mating, Yeast two-hybrid array, Protein-protein interactionThe rapidly growing knowledge of protein-protein interaction networks (interactomes) for model organisms and human provides a network-based model to understand molecular and cellular biology. Recently, virus-host relationships also began to be studied at the proteome level by identifying interactions between viral and host-cell proteins (1-5), as reviewed in ref. 6. These virus-host interactomes compose a repertoire of interactions between viral and human proteins that can be analyzed in a network approach. By this mean, viral infections can be viewed as the expression of new constraints imposed by the virus on the cellular interactome.

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“…AD cDNAs were amplified by PCR from zymolase-treated yeast colonies using primers that hybridized within the pPC86 regions flanking cDNA inserts. PCR products were sequenced, and cellular interactors were identified by multiparallel BLAST analysis (39).…”

Section: Methodsmentioning

confidence: 99%

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

Bouraï

Lucas‐Hourani

Gad

et al. 2012

J Virol

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102

111

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h 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 twohybrid 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.

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pISTil: a pipeline for yeast two-hybrid Interaction Sequence Tags identification and analysis

Cited by 11 publications

References 18 publications

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Virus–Human Cell Interactomes

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

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