scholarly journals Identification of the RNA-Binding, Dimerization, and eIF4GI-Binding Domains of Rotavirus Nonstructural Protein NSP3

1999 ◽  
Vol 73 (7) ◽  
pp. 5411-5421 ◽  
Author(s):  
Maria Piron ◽  
Thierry Delaunay ◽  
Jeanne Grosclaude ◽  
Didier Poncet
Keyword(s):  
Amino Acids ◽  
Hybrid System ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Domain ◽  
Initiation Factor ◽  
Yeast Two Hybrid ◽  
Dimerization Domain ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3′ end of the rotavirus mRNAs. NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein. Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149. Similar results were obtained with NSP3 from group C rotaviruses. Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding. The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system. The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system. NSP3 is composed of two functional domains separated by a dimerization domain.

scholarly journals Rice Grassy Stunt Tenuivirus Nonstructural Protein p5 Interacts with Itself To Form Oligomeric Complexes In Vitro and In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 769-775 ◽  
Author(s):  
Pritsana Chomchan ◽  
Shi-Fang Li ◽  
Yukio Shirako
Keyword(s):  
Hybrid System ◽  
Nonstructural Protein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Western Blots ◽  
Yeast Two Hybrid System ◽  
Rice Tissue ◽  
Two Hybrid

ABSTRACT We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).

scholarly journals Complete Protein Linkage Map of Poliovirus P3 Proteins: Interaction of Polymerase 3Dpol with VPg and with Genetic Variants of 3AB

1998 ◽  
Vol 72 (8) ◽  
pp. 6732-6741 ◽  
Author(s):  
Wenkai Xiang ◽  
Andrea Cuconati ◽  
Debra Hope ◽  
Karla Kirkegaard ◽  
Eckard Wimmer
Keyword(s):  
Hybrid System ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Yeast Cells ◽  
Single Amino Acid ◽  
Genome Replication ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT Poliovirus has evolved to maximize its genomic information by producing multifunctional viral proteins. The P3 nonstructural proteins harbor various activities when paired with different binding partners. These viral polypeptides regulate host cell macromolecular synthesis and function as proteinases, as RNA binding proteins, or as RNA-dependent RNA polymerase. A cleavage product of the P3 region is the genome-linked protein VPg that is essential in the initiation of RNA synthesis. We have used an inducible yeast two-hybrid system to analyze directly protein-protein interactions among P3 proteins. Sixteen signals of homo- or heterodimer interactions have been observed and have been divided into three groups. Of interest is the newly discovered affinity of VPg to 3Dpol that suggests direct interaction between these molecules in genome replication. A battery of 3AB variants (eight clustered-charge-to-alanine changes and five single-amino-acid mutations) has been used to map the binding determinants of 3AB-3AB interaction which were found to differ from the amino acids critical for the 3AB-3Dpol interaction. The viral proteinase 3Cpro was not found to interact with other 3Cpro molecules or with any other P3 polypeptide in yeast cells, a result confirmed by glutaraldehyde cross-linking. The weak apparent interaction between 3AB and 3CDpro scored in the yeast two-hybrid system was in contrast to a strong signal by far-Western blotting. The results elucidate, in part, previous results of biochemical and genetic analyses. The role of the interactions in RNA replication is addressed.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid
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