Analysis of the interaction of viral RNA replication proteins by using the yeast two-hybrid assay.
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.
O'Reilly
E K
EK
Department of Biology, Indiana University, Bloomington 47405, USA.
Paul
J D
JD
Kao
C C
CC
D017795:Bromovirus / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000254:growth & development; D017384:Sequence Deletion; D015533:Transcriptional Activation; D014779:Virus Replication
D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D020365:RNA Helicases