Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Abstract

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 × 10 6 (RNA 1) and 0.5 × 10 6 (RNA 2), respectively. Virions contained two proteins, β with a molecular weight of 43,000 (43K) and γ (5K), and traces of a third protein, α (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein α. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A , α, β, γ, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [ 35 S]methionine-labeled cultures. All but protein γ could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein α and the coordinate appearance of proteins β and γ, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor α. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and α, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor α peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.