Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, 32610-0266, USA.
Virology. 2010 May 25;401(1):49-60. doi: 10.1016/j.virol.2010.01.020. Epub 2010 Mar 5.
The vaccinia H5 protein has been implicated in several steps of virus replication including DNA synthesis, postreplicative gene transcription, and virion morphogenesis. Our recent mapping of mutants in the consolidated Condit-Dales collection identified a temperature-sensitive vaccinia mutant in the H5R gene (Dts57). We demonstrate here that Dts57 has a DNA negative phenotype, strongly suggesting a direct role for H5 in DNA replication. We used a temperature shift protocol to determine the impact of H5 temperature sensitivity on postreplicative gene expression and observed changes in the pattern of postreplicative viral mRNA metabolism consistent with a role of H5 in postreplicative transcription. Finally, using a rifampicin release temperature shift protocol, we show that H5 is involved in multiple steps of virion morphogenesis. These data demonstrate directly that H5 plays roles in DNA replication, transcription and morphogenesis in vivo.
痘苗 H5 蛋白已被牵涉到包括 DNA 合成、复制后基因转录和病毒形态发生在内的病毒复制的几个步骤中。我们最近在整合的 Condit-Dales 突变体库中定位突变体时，在 H5R 基因（Dts57）中发现了一个温度敏感型痘苗突变体。我们在此证明 Dts57 具有 DNA 阴性表型，这强烈表明 H5 在 DNA 复制中具有直接作用。我们使用温度转换方案来确定 H5 温度敏感性对复制后基因表达的影响，并观察到与 H5 在复制后转录中作用一致的复制后病毒 mRNA 代谢模式的变化。最后，使用利福平释放温度转换方案，我们表明 H5 参与病毒形态发生的多个步骤。这些数据直接证明 H5 在体内复制、转录和形态发生中发挥作用。
