He B, Chou J, Brandimarti R, Mohr I, Gluzman Y, Roizman B
The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Illinois 60637, USA.
J Virol. 1997 Aug;71(8):6049-54. doi: 10.1128/JVI.71.8.6049-6054.1997.
Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the alpha subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral gamma(1)34.5 gene, eIF-2alpha is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with gamma(1)34.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the alpha47 gene and junction with the inverted repeats flanking the unique short (U(S)) sequence of the HSV-1 DNA resulting in the juxtaposition of the alpha47 promoter to the coding domain of the U(S)11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the gamma(1)34.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the alpha47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of alpha47 gene sequences, (ii) accumulated U(S)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, gamma(1)34.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the gamma(1)34.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2alpha by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.
早期研究表明，单纯疱疹病毒1型（HSV-1）感染人类细胞会导致RNA依赖性蛋白激酶（PKR）激活，但真核起始因子2（eIF-2）的α亚基未被磷酸化，且蛋白质合成未受影响。在缺乏病毒γ(1)34.5基因的情况下，eIF-2α被磷酸化，蛋白质合成提前终止（J. Chou、J. J. Chen、M. Gross和B. Roizman，《美国国家科学院院刊》92:10516 - 10520，1995年）。最近的另一篇论文报道了对第二位点抑制突变体的筛选，这些突变体在感染γ(1)34.5 - 突变体的细胞中表现出接近野生型的蛋白质合成（I. Mohr和Y. Gluzman，《欧洲分子生物学组织杂志》15:4759 - 4766，1996年）。在此，我们报道了自发的HSV-1抑制突变体Sup-1的特性，其特征是自发缺失503 bp，该区域包含α47基因的结构域以及与HSV-1 DNA独特短序列（U(S)）侧翼反向重复序列的连接处，导致α47启动子与U(S)11基因的编码结构域并列。该突变体不表现出γ(1)34.5 - 病毒特有的蛋白质合成终止现象。具体而言，SK-N-SH人神经母细胞瘤细胞中的Sup-1：（i）未表现出α47基因的功能，其特征是与α47基因序列缺失一致，经通透处理的细胞中肽跨内质网的转运减少；（ii）积累的U(S)11蛋白水平与野生型亲本类似，但该蛋白在感染后的较早时间产生，正如启动子变化所预期的那样；（iii）像亲本γ(1)34.5 - 病毒一样激活PKR，但（iv）不会导致蛋白质合成提前终止，因此与野生型亲本病毒相似，而非与其衍生的γ(1)34.5 - 病毒相似。我们得出结论，Sup-1阻断与激活的PKR使eIF-2α磷酸化相关的蛋白质合成终止的机制，难以用以缺失为特征的二次突变来解释。
