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PMID: 9311832 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.

Journal of virology ·Vol. 71 ·No. 10 ·1997-10-00 ·Pages 7526-32

O'Reilly EK, Paul JD, Kao CC

Abstract

The yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. However, the biological relevance of these two-hybrid interactions to viral positive-strand RNA replication has not been demonstrated. The brome mosaic virus (BMV) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the BMV 1a helicase-like and 2a polymerase-like proteins. We have tested wild-type 1a and 18 insertion mutations of 1a and found a perfect correlation between the in planta phenotypes and their ability to interact with 2a in the two-hybrid system. This finding allowed further characterization of the interaction between and among the BMV viral proteins. Using the two-hybrid assay, we have found that the interaction between the helicase-like region of 1a and the N terminus of 2a is stabilized by the presence of the centrally conserved polymerase-like domain of 2a. We have also identified a novel interaction between the 1a helicase-like protein and itself. Additionally, we have found this interaction in two related tripartite RNA viruses, cowpea chlorotic mottle virus and cucumber mosaic virus. We have demonstrated that this protein-protein interaction is specific to homologous pairings of the protein.

MeSH Terms
Bromovirus/genetics,physiology Cloning, Molecular/methods DNA-Directed RNA Polymerases/metabolism Mutagenesis, Insertional Mutagenesis, Site-Directed Oligodeoxyribonucleotides Plasmids Polymerase Chain Reaction RNA Helicases RNA Nucleotidyltransferases/metabolism RNA, Viral/biosynthesis Saccharomyces cerevisiae/growth & development Sequence Deletion Transcriptional Activation Virus Replication
Chemicals
Oligodeoxyribonucleotides RNA, Viral RNA Nucleotidyltransferases DNA-Directed RNA Polymerases RNA Helicases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
O'Reilly E K
Department of Biology, Indiana University, Bloomington 47405, USA.
Paul J D
Kao C C
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.71.10.7526-7532.1997
Published
1997-10-00
Pages
7526-32
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC192099
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.71.10.7526-7532.1997 PMC Full Text View journal details
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