Bacteriophage theta6 contains three pieces of double-stranded RNA (S, M and L) in its nucleocapsid. The pieces possess different genetic information but have similar termini. The packaging of the genomic segments is precise in that virions contain one each of the three normal segments.
The aim of this proposal is to determine how the packing system is able to select one of each of the chromosomes and how the system acts to replicate the genome. The entire genome of theta6 has been cloned as cDNA and completely sequenced. Procapsids are produced from cDNA copies of segment L in both Escherichia coli and Pseudomonas phaseolicola, the natural host of theta6. These procapsids care capable of packaging single stranded viral RNA, synthesizing the complementary minus strand to form double stranded RNA and then transcribing this dsRNA to form plus ssRNA. Procapsids will be isolated and used to study both the replicase and packaging reactions. RNA transcripts made from cDNA constructions of the genomic segments can be packaged by the procapsids. The regions of the RNA involved with packaging will be manipulated so as to clarify the interaction between the RNA and the packaging and replication machinery. We will test a new model of packaging that involves retention of the correct complement of genomic segments by the formation of a ternary complex inside the procapsid after non selective entry. A system has been developed that facilitates the isolation and analysis of mutants in three of the replicase proteins. We will use this system to define the domains in proteins P2, P4 and P7 that are involved in packaging, minus and plus strand synthesis. The nature of the mechanisms of the mechanisms of selective packaging in viruses of segmented genomes is currently unknown. We have developed a novel model that explains the specific packaging. The elucidation of the mechanism for theta6 has relevance to the packaging of other segmented- genome viruses. Several members of the reoviridae, e.g. rotavirus and blue tongue virus, are the etiologic agents of important human and animal diseases.
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