Poon A P, Roizman B
Marjorie B. Kovler Viral Oncology Laboratory, University of Chicago, Illinois 60637, USA.
J Virol. 1995 Dec;69(12):7658-67. doi: 10.1128/JVI.69.12.7658-7667.1995.
alpha trans-inducing factor (alpha TIF, VP16, Vmw65) is an essential structural protein of herpes simplex virus, being required for virion assembly. The protein also forms complexes with host proteins and a response element and transactivates the alpha genes which carry this element. The protein contains an acidic carboxyl terminus required for transactivation and a much larger amino-terminal domain required for promoter recognition. We report the first set of temperature-sensitive (ts) mutations deliberately introduced into the protein by substitution of the cysteine codons with those specifying glycine at positions 78, 102, and 176, either singly or in combinations. We report the following results. (i) All mutated proteins synthesized in vitro formed complexes with the DNA response element at room temperature. However, the mutant with the triple substitution and two mutants with substitutions in two of the three cysteines exhibited a ts phenotype at 33 and 37 degrees C, and one exhibited a ts phenotype only at 37 degrees C. (ii) Replacement of wild-type alpha TIF with genes carrying substitutions in any two cysteines conferred a ts phenotype for replication at 39.5 degrees C. Shift-down experiments indicated that the 10(4)- to 10(5)-fold reduction in virus yield at the nonpermissive temperature was due to the disfunction of alpha TIF late in infection, presumably in virion maturation. (iii) The alpha TIF expressed in cells infected with mutant viruses exhibited the same ts phenotype in protein-DNA complex formation as those expressed in vitro from mutated plasmids. Although the virus carrying the alpha TIF substitutions at Cys-102 and Cys-176 failed to induce a reporter gene linked to the alpha 4 promoter at 39.5 degrees C, it replicated as well as the parent virus in cells maintained for the first 10 h of infection at 39.5 degrees C. We conclude the following. (i) Formation of DNA-protein complexes containing alpha TIF is a poor prognosticator of alpha TIF function. (ii) The data presented here and in the literature strongly support the hypothesis that the secondary structure of the alpha TIF is very sensitive to deletions or insertions which probably affect the interaction of alpha TIF with both viral proteins in the virion and cellular proteins during infection. As a consequence, deletion-insertion mutagenesis may not shed useful information on the role of transactivating function of alpha TIF in infection. (iii) Since cysteines may play a role in stabilizing the secondary structure of proteins, substitutions of cysteines may be a powerful technique for site-specific construction of ts mutants in essential viral proteins.
α反式诱导因子（αTIF、VP16、Vmw65）是单纯疱疹病毒的一种必需结构蛋白，参与病毒体组装。该蛋白还与宿主蛋白及一个反应元件形成复合物，并反式激活携带此元件的α基因。该蛋白含有反式激活所需的酸性羧基末端以及启动子识别所需的大得多的氨基末端结构域。我们报道了通过将78、102和176位的半胱氨酸密码子分别或组合替换为甘氨酸密码子，首次特意引入该蛋白的一组温度敏感（ts）突变。我们报道了以下结果。（i）所有体外合成的突变蛋白在室温下均与DNA反应元件形成复合物。然而，三重替换突变体以及三个半胱氨酸中有两个被替换的两个突变体在33℃和37℃时表现出ts表型，一个仅在37℃时表现出ts表型。（ii）用在任意两个半胱氨酸处有替换的基因取代野生型αTIF，赋予了在39.5℃复制的ts表型。温度下调实验表明，在非允许温度下病毒产量降低10⁴至10⁵倍是由于感染后期αTIF功能失调，推测是在病毒体成熟过程中。（iii）感染突变病毒的细胞中表达的αTIF在蛋白质-DNA复合物形成中表现出与从突变质粒体外表达的αTIF相同的ts表型。尽管携带Cys-102和Cys-176处αTIF替换的病毒在39.5℃时未能诱导与α4启动子相连的报告基因，但它在感染的前10小时维持在39.5℃的细胞中与亲本病毒一样复制。我们得出以下结论。（i）含有αTIF的DNA-蛋白质复合物的形成并不能很好地预测αTIF的功能。（ii）此处及文献中的数据有力地支持了以下假设：αTIF的二级结构对缺失或插入非常敏感，这可能会影响感染期间αTIF与病毒体中的病毒蛋白以及细胞蛋白的相互作用。因此，缺失-插入诱变可能无法提供关于αTIF反式激活功能在感染中的作用的有用信息。（iii）由于半胱氨酸可能在稳定蛋白质二级结构中起作用，半胱氨酸的替换可能是在必需病毒蛋白中进行位点特异性构建ts突变体的有力技术。
