Immunological Analysis of Brome Mosaic Virus Replicase

Summary Antisera specific for the non-structural proteins 1a and 2a of brome mosaic virus (BMV) were prepared by the gene fusion method. Plasmids were constructed which expressed parts of 1a and 2a in Escherichia coli as fusion proteins with Protein A. After induction of fusion protein synthesis in E. coli, the fusion proteins accumulated in insoluble fractions. Antisera raised against these proteins (anti-1a and anti-2a sera) reacted specifically with the respective proteins translated in vitro and in vivo. These antisera were used to investigate the involvement of the 1a and 2a proteins in the BMV replicase preparation which initiated complementary strand synthesis de novo on BMV RNA. Immunoblot analysis using these antisera revealed the presence of 1a and 2a in the BMV replicase fraction. The replicase activity was inhibited by the addition of anti-1a serum, but not anti-2a serum. Further addition of Protein A-Sepharose to remove each immunocomplex gave similar results. These results suggested the involvement of at least the 1a protein in our BMV replicase preparation.