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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.

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PurposeThe aim of this study was to evaluate the salvage radiotherapy outcome in patients with local recurrent esophageal cancer after radical radiochemotherapy (RCT).MethodsA total of 114 patients with local recurrent esophageal squamous cell carcinoma after initial radical RCT were retrospectively analyzed. Fifty-five (55) patients belonged to the salvage radiotherapy group (SR group) and 59 patients to the non-salvage radiotherapy group (NSR group).ResultsThe median survival time after-recurrence was 4 months in all patients. The 1, 2, 3 year overall survival (OS) rates were 83.6%, 41.8% and 21.8% respectively in the SR group, and 57.6%, 16.9%, and 8.5% in the NSR group. The 6-month and 1-year survival rates after-recurrence were 41.8% and 16.4% respectively in the SR group, and 11.9% and 3.4% respectively in the NSR group. A salvage radiation dose > 50 Gy after initial radical RCT, improved the survival of patients with local recurrent esophageal cancer. Three patients (5.45%) from the SR group showed more than 3-grade radiation pneumonitis. In addition, esophageal fistula/perforation was observed in 11 cases (20.0%) in the SR group and in 8 cases (13.6%) in the NSR group.ConclusionsSalvage treatment after definitive RCT may improve the overall survival and survival after-recurrence of patients with local recurrent esophageal cancer.

A model that explains the stoichiometric packaging of the chromosomes of ⌽6, a bacteriophage with a genome of three unique double-stranded RNA segments, is proposed and supported. Ordered switches in packaging specificity and RNA synthesis are determined by the amount of RNA within the procapsid. The plus strand of segment S binds to one of several sites on the outside of the empty procapsid. The RNA enters and the procapsid expands so that the S sites are lost and M sites appear. Packaging of segment M results in the loss of the M sites and the appearance of the L sites. Packaging of L readies the particle for minus-strand synthesis. If any of the segments is less than normal size, packaging of that class of segments continues until the normal content of RNA for that segment is packaged and the binding sites then change.

Eight different bacteriophages were isolated from leaves ofPisum sativum, Phaseolus vulgaris,Lycopersicon esculentum, Daucus carota sativum,Raphanus sativum, and Ocimum basilicum. All contain three segments of double-stranded RNA and have genomic-segment sizes that are similar but not identical to those of previously described bacteriophage φ6. All appear to have lipid-containing membranes. The base sequences of some of the viruses are very similar but not identical to those of φ6. Three of the viruses have little or no base sequence identity to φ6. Two of the viruses, φ8 and φ12, contain proteins with a size distribution very different from that of φ6 and do not package genomic segments of φ6. Whereas φ6 attaches to host cells by means of a pilus, several of the new isolates attach directly to the outer membrane. Although the normal hosts of these viruses seem to be pseudomonads, those viruses that attach directly to the outer membrane can establish carrier states in Escherichia coli or Salmonella typhimurium. One of the isolates, φ8, can form plaques on heptoseless strains of S. typhimurium.

BackgroundBacteriophage Φ12 is a member of the Cystoviridae and is distinct from Φ6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Φ12 in the amino acid sequences of several proteins. Bacteriophage Φ2954 is a member of this group.ResultsΦ2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Φ12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Φ2954 show no similarity to those of Φ12, segment M of Φ2954 could be acquired by Φ12 resulting in a change of host specificity.ConclusionsWe have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression.