The reconstitution of infective bacteriophage R17.

Bacteriophage R17 contains two major components: a single RNA molecule of molecular weight 1.1 X 106,1 and approximately 180 identical coat protein subunits of molecular weight 14,000.2 This apparently simple structure invites attempts to reassemble the phage from isolated RNA and coat protein in analogy with the classic reconstitution of tobacco mosaic virus.3 Such reconstitution experiments have been performed with the closely related phages M\S24 and fr,5 yielding particles which appeared similar to normal bacteriophage in electron micrographs. However, the MS2 particles had no significant infectivity, and the fr assembly produced only a slight increase in infectivity over the isolated components. In addition, the assembled particles in both cases sedimented at 70S in sucrose density gradients, whereas normal phage sediments at 80S. The failure to achieve significant infectivity in these reconstitution experiments was not surprising in view of various genetic and physiological evidence which has long suggested that another structural component might be present in mature RNA phage particles. First, studies of RNA phage amber mutants have revealed that uninfective progeny particles are produced by phages whose genomes contain a mutation in the A cistron,6-8 which can be distinguished genetically from the cistrons encoding coat protein and the phage-specific RNA synthetase.9 10 Second, the use of polyacrylamide gel electrophoresis has both detected the protein encoded by the A cistron within phage-infected cells11 12 and provided hints that the A cistron product itself is present in small amounts in the virus particle.12 Aided by the fact that the A cistron protein contains histidine, whereas the coat protein does not, one of us has shown recently'3 that it is present in wild-type R17 and absent from the defective particles produced by A cistron amber mutants. These experiments, and the fact that both the defective particles'4 and the reassembled particles of MS2 and fr sediment at 70S, suggested that the uninfective reconstituted particles might differ from normal bacteriophage by lacking the A protein.45 Subsequent work has now led to the purification of the A protein.13 It has a molecular weight of approximately 35,000 and is present probably in only one copy per phage particle. It is highly insoluble in aqueous buffers and can be obtained at high concentrations only in denaturing solvents. Possession of the A protein provides the opportunity to perform reconstitution experiments with all the known components of an RNA bacteriophage present in the assembly mixture. Here we report such experiments in which a significant synthesis of infective particles occurs only when isolated A protein is added to the assembly mixture. Materials and Methods. -Phage and bacterial strains: Bacteriophage R17, and S26, the Hfr bacteria on which it was grown, were described previously. '0 Infectivity assays were performed on S26 or on S26RIE, a strain isogenic with S26 but containing the amber suppressor SUI. " The coat protein amber mutant amB2 was isolated by G. Gussin. 16' ' Purification of bacteriophage: R17 was purified according to the method of Gesteland and Boedtker, ' with the addition of a second CsCl banding step.