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Isolation of a mutant that changes genomic packaging specificity in phi6
1Department of Microbiology, The Public Health Research Institute, 455 First Avenue, New York, New York, 10016, USA.
Abstract:
Bacteriophage phi6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Plus strand transcripts of the segments are packaged in a serially dependent fashion in which S can package alone, M depends on S, and L depends on S and M. We have isolated a mutant form of the virus in the carrier state that has lost segment S. This finding presented an apparent anomaly with respect to the packaging program. Sequencing of gene 1 of segment L in this virus showed a translational change of arginine to glycine at the 14th position. Procapsids prepared from cDNA containing this mutation show behavior in in vitro packaging that is consistent with the phenotype of the mutant virus. The procapsids are able to package segment S alone, but this RNA is present in reduced amounts when the other segments are present. Segments M and L package without dependence on segment S. The mutant virus appears to produce procapsids that are at the second stage of the packaging program.
Insights
A bacteriophage phi6 mutant lacking segment S was identified. This virus exhibits altered RNA packaging, suggesting procapsids may adapt to different packaging stages.
Area of Science:
- Virology
- Molecular Biology
- Structural Biology
Background:
- Bacteriophage phi6 possesses a three-segment double-stranded RNA genome within a polyhedral procapsid.
- RNA packaging follows a strict serial dependence: S, then M, then L.
Purpose of the Study:
- Investigate an anomalous bacteriophage phi6 mutant that lost its S segment.
- Characterize the packaging behavior and underlying molecular changes in this mutant.
Main Methods:
- Isolation and characterization of a bacteriophage phi6 mutant.
- Sequencing of gene 1 in segment L to identify mutations.
- In vitro packaging assays using procapsids from mutant cDNA.
Main Results:
- The mutant showed a translational change (arginine to glycine) at position 14 in gene 1 of segment L.
- In vitro packaging revealed procapsids could package S alone, but with reduced efficiency when M and L were present.
- Segments M and L packaging became independent of segment S.
Conclusions:
- The identified mutation in segment L alters procapsid structure and function.
- Mutant procapsids exhibit a modified packaging program, potentially representing an intermediate stage.
- This study provides insights into the flexibility and regulation of bacteriophage RNA packaging.