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 la and the polymera se-like 2a proteins. One hundred fifteen amino acids of the 2a N-termi nus 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 la helicase-like domain suggests that higher order structures mig ht be necessary for the protein's interaction with 2a. To explore the structural properties of la, we used limited proteolysis of in vitro-t ranslated la protein. Treatment of 1a and its deletion derivatives wit h papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50-60 kDa is highly resistant under our assay condit ions to proteolysis, while the N-terminus is rapidly degraded. All tes ted mutations in the helicase-like region that renders this region pro tease-sensitive have previously been found to be defective for RNA rep lication in vivo. To complement the in vitro studies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in ye ast using the two-hybrid system. Mutations previously known to disrupt 1a-2a interaction also prevented interaction in yeast. Furthermore, r esults from two-hybrid analysis suggest that the structural domain map ped in vitro is important for 1a-2a interaction. Finally, we found tha t the helicase-like proteins of three other tripartite RNA Viruses als o contain equivalently located protease-resistant domains. (C) 1995 Ac ademic Press, Inc.