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A system is described for assembling infectious bacteriophage 46 nucleocapsids in vitro. Procapsids encoded by cDNA copies of genomic segment L in Escherichia coli were used to package and replicate viral RNA segments.The resulting filed particles were shown to be capable of infecting host cell spheroplasts after incubation with purified nucleocapsid shell protein P8. The infected spheroplasts yielded infectious virions. A modified cDNA-derived RNA segment was inserted into virions by this method. The resulting infectious virions contained the same 4-base-pair deletion as the modified cDNA. These findings support the contention that the preformed procapsids are the "machine" that replicates the 46 genome, by showing that the cDNA-derived procapsids are competent to package and replicate RNA properly.Bacteriophage 46 is a double-stranded RNA (dsRNA) virus of Pseudomonas phaseolicola (1). Its segmented genome, consisting of three separate pieces (2, 3), is associated with a complex isometric nucleocapsid (NC) structure (4, 5). The NC has five protein components, one of which (protein P8) forms the outer shell of the particle (4, 5). The other four proteins (P1, P2, P4, and P7) comprise an inner particle that displays the viral transcriptase and replicase activities (6-10). The 06 NC is enclosed in a lipid-protein envelope, the components of which have vital functions in the initial steps of the phage infection (1,(11)(12)(13).The internal NC proteins, P1, P2, P4, and P7, are encoded by the large (L) genomic segment (14, 15) and are synthesized early in 06 infection (5). Particles (procapsids) consisting of these four proteins and displaying RNA polymerase activity can be purified from Escherichia coli or P. phaseolicola cells harboring cDNA copies of the L segment in an expression vector (9, 10). Protein P1 forms the dodecahedral framework of the procapsids, to which the other proteins are attached (9,17,18). Defective particles lacking protein P2 have no polymerase activity, suggesting that this protein forms the active site of the enzyme. The cDNA-derived procapsids are capable of packaging and replicating viral (+)-strand RNA to double-stranded genomic segments, as well as of producing transcripts using the dsRNA formed as template (10).Recently, a technique for infecting P. phaseolicola spheroplasts with the purified 06 NC has been developed (19). Bacteriophage 06 (1) was used for the preparation of phage NCs used in single-stranded RNA (ssRNA) production and as the source of the NC coat protein, P8.The expression plasmid pLM450 (10) harboring 06 L-segment cDNA was used for the production of phage procapsids in E. coli JM109. Strain JM109 was the host for the propagation of recombinant plasmids. E. coli strain CJ236 is a dut, ung derivative that is used for the generation of uracilcontaining DNA in phagemids (21 tTo whom reprint requests should be addressed. 9173The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertiseme...

The genome of the lipid-containing bacteriophage +6 contains three segments of double-stranded RNA (dsRNA). We prepared cDNA copies of the viral genome and cloned this material in plasmids that replicate in Escherichia coli and Pseudomonas phaseolicola, the natural host of 406. These plasmids direct the formation of viral proteins and the assembly of structures similar to viral procapsids containing proteins P1, P2, P4, and P7. We found that these particles are capable of taking up viral single-stranded RNA and synthesizing the minus strands to produce dsRNA structures. Once the dsRNA is formed, it is then used as a template for the production of viral plus strands in a reaction that resembles normal transcription. The particles were also capable of directly transcribing exogenous dsRNA. The replicase reactions were specific for 46 RNA, were specific for procapsids, and resulted in substantial incorporation of product dsRNA into particles. These results offer strong support to a model in which genomic packaging is done by preformed procapsids.

The RNA-dependent RNA polymerases (RdRPs) of Cystoviridae bacteriophages, like those of eukaryotic viruses of the Reoviridae, function inside the inner capsid shell in both replication and transcription. In bacteriophage ⌽6, this inner shell is first assembled as an icosahedral procapsid with recessed 5-fold vertices that subsequently undergoes major structural changes during maturation. The tripartite genome is packaged as singlestranded RNA molecules via channels on the 5-fold vertices, and transcripts probably exit the mature capsid by the same route. The RdRP (protein P2) is assembled within the procapsid, and it was thought that it should be located on the 5-fold axes near the RNA entry and exit channels. To determine the initial location of the RdRP inside the procapsid of bacteriophage ⌽6, we performed cryo-electron microscopy of wild type and mutant procapsids and complemented these data with biochemical determinations of copy numbers. We observe ring-like densities on the 3-fold axes that are strong in a mutant that has ϳ10 copies of P2 per particle; faint in wild type, reflecting the lower copy number of ϳ3; and completely absent in a P2-null mutant. The dimensions and shapes of these densities match those of the known crystal structure of the P2 monomer. We propose that, during maturation, the P2 molecules rotate to occupy positions closer to adjacent 5-fold vertices where they conduct replication and transcription.

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We investigated the protein composition of the lipid-containing bacteriophage 06. We also studied the synthesis of phage-specific proteins in the host bacterium Pseudomonas phaseolicola HB1OY. The virion was found to contain 10 proteins of the following molecular weights: P1,