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The cystoviridae are bacteriophages with genomes of three segments of dsRNA enclosed within a polyhedral capsid. Two members of this family, Phi6 and Phi8, have been shown to form carrier states in which the virus replicates as a stable episome in the host bacterium while expressing reporter genes such as kanamycin resistance or lacalpha. The carrier state does not require the activity of all the genes necessary for phage production. It is possible to generate carrier states by infecting cells with virus or by electroporating nonreplicating plasmids containing cDNA copies of the viral genomes into the host cells. We have found that carrier states in both Phi6 and Phi8 can be formed at high frequency with all three genomic segments or with only the large and small segments. The large genomic segment codes for the proteins that constitute the inner core of the virus, which is the structure responsible for the packaging and replication of the genome. In Phi6, a carrier state can be formed with the large and middle segment if mutations occur in the gene for the major structural protein of the inner core. In Phi8, carrier state formation requires the activity of genes 8 and 12 of segment S.

The inner core of bacteriophage phi8 is capable of packaging and replicating the plus strands of the RNA genomic segments of the virus in vitro. The particles composed of proteins P1, P2, P4, and P7 can be assembled in cells of E. coli that carry plasmids with cDNA copies of genomic segment L. The gene arrangement on segment L was found to differ from that of other cystoviruses in that the gene for the ortholog of protein P7 is located at the 3' end of the plus strand rather than near the 5' end. In place of the normal location of gene 7 is gene H, whose product is necessary for normal phage development, but not necessary for in vitro genomic packaging and replication. Genomic packaging is dependent upon the activity of an NTPase motor protein, P4. P4 was purified from cell extracts and was found to form hexamers with little NTPase activity until associated with inner core particles. Labeling studies of in vitro packaging of phi8 RNA do not show serial dependence; however, studies involving in vitro packaging for the formation of live virus indicate that packaging is stringent. Studies with the acquisition of chimeric segments in live virus indicate that phi8 does package RNA in the order s/m/l. The inner core of bacteriophage phi8 differs from that of its relatives in the Cystoviridae in that the major structural protein P1 is able to interact with the host cell membrane to effect penetration of the inner core into the cell.