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Construction of a transducing virus from double-stranded RNA bacteriophage phi6: establishment of carrier states in
S Onodera1, V M Olkkonen, P Gottlieb
1Department of Microbiology, Public Health Research Institute, New York, New York 10016.
Abstract:
Bacteriophage phi 6 contains three double-stranded RNA (dsRNA) genomic segments. We have constructed a plasmid that contains a cDNA copy of the middle (M) segment, with a gene for kanamycin resistance (kan) inserted into the PstI site. A transcript of this cDNA was incorporated in vitro into procapsids along with natural transcripts of the S and L segments. The procapsids were coated with nucleocapsid surface protein P8 and transfected into Pseudomonas syringae pv. phaseolicola. The resulting infectious virus, phi 6 K1, was found to contain an M segment that was 1.2 kbp larger than the normal 4.1 kbp. K1 formed small, turbid plaques, and its genome was unstable. Preparations of K1 contained from about 0.1 to 10% large, clear-plaque forms of the virus which were usually missing the kan gene, and in some cases, the resulting segment M was smaller than its normal size. Cells picked from lawns of host cells infected with K1 yielded colonies that were resistant to kanamycin (Kan). These colonies could be passaged on kanamycin-containing medium. The cells were found to contain large amounts of dsRNA corresponding to the viral genomic segments. Some strains continued to produce viable phage, while others lost this ability. One strain completely lost the small genomic segment S. Approximately 1 in 10,000 infected cells acquired the carrier state with the original phage isolate K1. However, we isolated a viral mutant that was able to induce the carrier state in 10 to 20% of the infected cells. The ability to use drug resistance as a test for the carrier state makes this system very useful for the study of the mechanisms of induction of persistent infections.
Insights
Researchers engineered bacteriophage phi 6 with a kanamycin resistance gene, creating a tool to study persistent infections. This modified phage helps investigate how bacteria develop drug resistance and carrier states during viral infections.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Bacteriophage phi 6 possesses a three-segment double-stranded RNA (dsRNA) genome.
- Understanding viral genome manipulation is crucial for studying host-pathogen interactions.
Purpose of the Study:
- To construct a modified bacteriophage phi 6 with a selectable marker for studying persistent infections.
- To investigate the mechanisms underlying the induction of carrier states in Pseudomonas syringae pv. phaseolicola.
Main Methods:
- A plasmid containing a cDNA copy of the bacteriophage phi 6 M segment with a kanamycin resistance gene was created.
- In vitro transcription and incorporation into procapsids, followed by transfection into host cells.
- Analysis of viral progeny, plaque morphology, genome stability, and host cell carrier state induction.
Main Results:
- The engineered phage phi 6 K1 exhibited an enlarged M segment and genome instability, producing kanamycin-resistant variants.
- Infected host cells developed a carrier state, retaining dsRNA and exhibiting variable phage production or loss of genomic segments.
- A specific viral mutant significantly increased the frequency of carrier state induction.
Conclusions:
- The engineered bacteriophage phi 6 system provides a valuable tool for studying persistent infections and carrier states.
- Drug resistance serves as an effective marker for identifying and analyzing the mechanisms of persistent viral infections.
- This research opens avenues for exploring viral persistence and host-pathogen dynamics in bacterial systems.