Rice S A, Klessig D F
J Virol. 1984 Jan;49(1):35-49. doi: 10.1128/JVI.49.1.35-49.1984.
The adenovirus type 2 (Ad2) host range mutant Ad2hr400 grows efficiently in cultured monkey cells at 37 degrees C, but is cold sensitive for plaque formation and late gene expression at 32.5 degrees C. After nitrous acid mutagenesis of an Ad2hr400 stock, cold-resistant variants were selected in CV1 monkey cells at 32.5 degrees C. One such variant, Ad2ts400, was also temperature sensitive (ts) for growth in both CV1 and HeLa cells. Marker rescue analysis has been used to show that the two phenotypes, cold resistant and temperature sensitive, are due to two independent mutations, each of which resides in a different segment of the gene encoding the 72-kilodalton DNA binding protein (DBP). The cold-resistant mutation (map coordinates 63.6 to 66) is a host range alteration that enhances the ability of the virus to express late genes and grow productively in monkey cells at 32.5 degrees C. The temperature-sensitive mutation is in the same complementation group and maps to the same segment of the DBP gene (map coordinates 61.3 to 63.6) as the well-characterized DBP mutant Ad5ts125. Like Ad5ts125, Ad2ts400 is unable to replicate viral DNA or to properly shut off early mRNA expression at the nonpermissive temperature. Two sets of experiments with Ad2ts400 suggest that DBP contains separate functional domains. First, when CV1 cells are coinfected at the nonpermissive temperature with Ad2 plus Ad2ts400 (Ad2 allows DNA replication and entry into, but not completion of, the late phase of infection), normal late gene expression and productive growth occur. Second, temperature shift experiments show that, although DNA replication is severely restricted at the nonpermissive temperature in ts400-infected monkey cells, late gene expression occurs normally. These results indicate that the DBP activity required for normal late gene expression in monkey cells is functional even when the DBP's DNA replication activity is disrupted.
腺病毒2型（Ad2）宿主范围突变体Ad2hr400在37℃时能在培养的猴细胞中高效生长，但在32.5℃时对蚀斑形成和晚期基因表达具有冷敏感性。在用亚硝酸诱变Ad2hr400储备液后，在32.5℃的CV1猴细胞中筛选出了抗冷变体。其中一个这样的变体Ad2ts400在CV1和HeLa细胞中的生长也具有温度敏感性（ts）。标记拯救分析已用于表明抗冷和温度敏感这两种表型是由两个独立的突变引起的，每个突变位于编码72千道尔顿DNA结合蛋白（DBP）的基因的不同区段。抗冷突变（图谱坐标63.6至66）是一种宿主范围改变，可增强病毒在32.5℃时在猴细胞中表达晚期基因和高效生长的能力。温度敏感突变与特征明确的DBP突变体Ad5ts125处于同一互补组，并定位到DBP基因的同一段（图谱坐标61.3至63.6）。与Ad5ts125一样，Ad2ts400在非允许温度下无法复制病毒DNA或正确关闭早期mRNA表达。用Ad2ts400进行的两组实验表明DBP包含独立的功能结构域。首先，当CV1细胞在非允许温度下与Ad2加Ad2ts400共感染时（Ad2允许DNA复制并进入但不能完成感染后期），正常的晚期基因表达和高效生长会发生。其次，温度转换实验表明，尽管在ts400感染的猴细胞中，在非允许温度下DNA复制受到严重限制，但晚期基因表达仍正常发生。这些结果表明，即使DBP的DNA复制活性被破坏，猴细胞中正常晚期基因表达所需的DBP活性仍然是有功能的。
