Hooda-Dhingra U, Thompson C L, Condit R C
Department of Biochemistry, State University of New York, Buffalo 14214.
J Virol. 1989 Feb;63(2):714-29. doi: 10.1128/JVI.63.2.714-729.1989.
We have carried out detailed phenotypic characterization of five temperature-sensitive (ts) mutants of vaccinia virus, the ts lesions of which have previously been mapped to two different subunits of the viral RNA polymerase. We have also attempted to determine the mechanism of temperature sensitivity in these mutants. Phenotypic characterization of each of the mutants showed that at the nonpermissive temperature, all five mutants exhibited normal levels of early viral mRNA and protein synthesis, but for an extended period of time, all mutants accumulated normal levels of DNA in abnormally large pools in the cell cytoplasm; all mutants were defective in the synthesis of late viral mRNA and proteins and in viral morphogenesis. In an attempt to address the mechanism of temperature sensitivity in these mutants, we measured the effect of a temperature shift on the ability of the mutants to direct late viral protein synthesis. If infected cells were shifted down from a nonpermissive temperature late during infection, late protein synthesis was initiated after a lag period of 1 to 2 h. If infected cells were shifted up from a permissive temperature early during infection, late protein synthesis continued to be defective. If infected cells were shifted up to the nonpermissive temperature after late protein synthesis had commenced, late protein synthesis was maintained at the nonpermissive temperature at the level observed when the temperature was shifted up. We interpret these results to mean that once a functional RNA polymerase has been assembled at the permissive temperature during a mutant infection, it remains functional at the nonpermissive temperature, but that the ts mutants are defective in the assembly of a newly synthesized RNA polymerase at the nonpermissive temperature. This interpretation implies that the virion RNA polymerase is responsible for early viral transcription and that a newly synthesized RNA polymerase transcribes late viral genes.
我们对痘苗病毒的五个温度敏感（ts）突变体进行了详细的表型特征分析，这些ts损伤先前已被定位到病毒RNA聚合酶的两个不同亚基上。我们还试图确定这些突变体中温度敏感性的机制。对每个突变体的表型特征分析表明，在非允许温度下，所有五个突变体早期病毒mRNA和蛋白质合成水平正常，但在较长一段时间内，所有突变体在细胞质中异常大的池中积累了正常水平的DNA；所有突变体在晚期病毒mRNA和蛋白质合成以及病毒形态发生方面均存在缺陷。为了探究这些突变体中温度敏感性的机制，我们测量了温度变化对突变体指导晚期病毒蛋白质合成能力的影响。如果在感染后期将感染细胞从非允许温度下调，晚期蛋白质合成会在1至2小时的延迟期后开始。如果在感染早期将感染细胞从允许温度上调，晚期蛋白质合成仍然存在缺陷。如果在晚期蛋白质合成开始后将感染细胞上调至非允许温度，晚期蛋白质合成会在非允许温度下维持在温度上调时观察到的水平。我们将这些结果解释为，在突变体感染期间，一旦在允许温度下组装了功能性RNA聚合酶，它在非允许温度下仍保持功能，但ts突变体在非允许温度下新合成RNA聚合酶的组装存在缺陷。这种解释意味着病毒粒子RNA聚合酶负责早期病毒转录，而新合成的RNA聚合酶转录晚期病毒基因。
