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 la helicase-like protein and itself, Additionally;, we hav 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 tile protein.