Mamoru Horikoshi

Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

In vitro, the coat protein of brome mosaic virus (BMV) inhibited RNA synthesis by replicase extracted from BMV-infected barley leaves. The inhibition was due to the interaction of coat protein with BMV RNA. Under the same conditions, no inhibition by TMV coat protein or bovine serum albumin was detected. For the complete inhibition of RNA synthesis in vitro, a coat protein:RNA ratio of 620:1 was required, and their preincubation before addition to the reaction mixture was essential. If the coat protein was added to the reaction mixture during incubation, synthesis continued for a few minutes at the level of the control (omission of coat protein), then decreased gradually, and stopped 6 min after the addition of coat protein. These results suggest that the inhibitory effect of coat protein on RNA synthesis in vitro is due to the interference with the binding site of replicase by partial reassembly of nucleoprotein and that this phenomenon may be a cause of cross protection.

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.