Biochemical and Genetic Analyses of the Interaction between the Helicase-like and Polymerase-like Proteins of the Brome Mosaic Virus

Replication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a–2a interaction. Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of 1a, we used limited proteolysis ofin vitro-translated 1a protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50–60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replicationin vivo.To complement thein vitrostudies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a–2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mappedin vitrois important for 1a–2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains.