Diaz-Guerra M, Esteban M
Department of Biochemistry, State University of New York, Health Science Center at Brooklyn 11203.
J Virol. 1993 Dec;67(12):7561-72. doi: 10.1128/JVI.67.12.7561-7572.1993.
We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
我们对感染了温度敏感型痘苗病毒突变体（ts36）的培养细胞中产生的病毒mRNA和蛋白质进行了详细分析。该突变体含有一种修饰的核苷三磷酸磷酸水解酶I（NPH-I），即一种依赖核酸的ATP酶。使用表达荧光素酶标记的重组病毒（ts36LUC），我们在七种不同的细胞系中表明，在非允许温度下，受体基因的早期表达受到强烈抑制（73.8%至98.7%）。不同早期病毒多肽的稳态水平也严重降低。对两个早期基因（DNA聚合酶和D5）的稳态mRNA水平分析表明，早期多肽合成的抑制与在非允许温度下积累的mRNA水平降低相关。对晚期病毒多肽和mRNA的稳态水平分析表明，NPH-I对中期和晚期基因表达的调节是直接的，而不仅仅是其抑制早期基因表达作用的结果。对拯救病毒（R36）的表征表明，ts36的温度敏感表型仅归因于NPH-I基因中的点突变。突变表型不是由于ts36病毒粒子中NPH-I水平降低，也不是由于该酶在非允许温度下感染的细胞中的稳定性差异，而是由于该蛋白质正常酶活性的抑制。在通透的纯化病毒粒子中测量病毒转录活性表明，痘苗病毒正常转录速率需要NPH-I。我们的研究结果表明ts36是痘苗病毒的一个严重缺陷的早期突变体，并证明NPH-I在控制病毒早期和晚期基因表达中起关键作用，可能是通过一种辅助功能来调节病毒生长周期中的mRNA转录。
