Sánchez A, Banerjee A K
Virology. 1985 Nov;147(1):177-86. doi: 10.1016/0042-6822(85)90237-5.
Cytoplasmic poly(A)-containing RNA from parainfluenza virus 3-infected cells was used as template to construct a cDNA library that was cloned into the EcoRV site of the plasmid pMG5. The resulting clones were screened with [32P]-labeled cDNA probes made from infected and mock-infected cell mRNAs. The virus specificity of the clones was confirmed by Northern blot hybridization. The viral clones were grouped into five different families by hybridization with individual size-selected reverse transcripts representing the major classes of poly(A)+-RNA from virus-infected cells. The five groups were shown to be unrelated on the basis of cross-colony hybridization and corresponded to five unique classes of intracellular viral poly(A)+-RNAs. Clones representing the NP and P genes of PIV-3 were identified by both hybrid-selected and hybrid-arrested translation. Clones specific for the P gene selected mRNA that directed the synthesis of P protein and another polypeptide of 21 kDa. This additional polypeptide comigrated with protein VP8 previously identified in virions and in infected cell lysates.
来自副流感病毒3感染细胞的细胞质含聚腺苷酸RNA被用作模板构建cDNA文库，该文库被克隆到质粒pMG5的EcoRV位点。用从感染和未感染细胞的mRNA制备的[32P]标记的cDNA探针筛选所得克隆。通过Northern印迹杂交确认克隆的病毒特异性。通过与代表病毒感染细胞中主要聚腺苷酸加RNA类别的个体大小选择的逆转录产物杂交，将病毒克隆分为五个不同的家族。基于菌落间杂交表明这五组是不相关的，并且对应于细胞内病毒聚腺苷酸加RNA的五个独特类别。通过杂交选择翻译和杂交捕获翻译鉴定了代表PIV-3的NP和P基因的克隆。针对P基因的克隆选择了指导P蛋白和另一种21 kDa多肽合成的mRNA。这种额外的多肽与先前在病毒粒子和感染细胞裂解物中鉴定的VP8蛋白迁移率相同。
