Onodera S, Olkkonen V M, Gottlieb P, Strassman J, Qiao X Y, Bamford D H, Mindich L
Department of Microbiology, Public Health Research Institute, New York, New York 10016.
J Virol. 1992 Jan;66(1):190-6. doi: 10.1128/JVI.66.1.190-196.1992.
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.
噬菌体φ6含有三个双链RNA（dsRNA）基因组片段。我们构建了一个质粒，其中包含中间（M）片段的cDNA拷贝，并在PstI位点插入了卡那霉素抗性（kan）基因。该cDNA的转录本与S和L片段的天然转录本一起在体外被包装进原衣壳。原衣壳被核衣壳表面蛋白P8包裹，并转染到菜豆假单胞菌中。所产生的感染性病毒φ6 K1，其M片段比正常的4.1 kbp大1.2 kbp。K1形成小的、浑浊的噬菌斑，其基因组不稳定。K1的制剂中含有约0.1%至10%的大的、清晰噬菌斑形式的病毒，这些病毒通常缺失kan基因，在某些情况下，产生的M片段比其正常大小小。从感染K1的宿主细胞菌苔中挑选的细胞产生了对卡那霉素（Kan）有抗性的菌落。这些菌落在含卡那霉素的培养基上可以传代。发现这些细胞含有大量与病毒基因组片段相对应的dsRNA。一些菌株继续产生有活力的噬菌体，而其他菌株则失去了这种能力。一个菌株完全失去了小的基因组片段S。大约每10000个感染细胞中有1个获得了与原始噬菌体分离株K1的携带状态。然而，我们分离出了一个病毒突变体，它能够在10%至20%的感染细胞中诱导携带状态。利用药物抗性作为携带状态的检测方法，使得这个系统对于研究持续性感染的诱导机制非常有用。
