Lengyel Joy, Guy Chandra, Leong Vivian, Borge Sarah, Rice Stephen A
Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
J Virol. 2002 Dec;76(23):11866-79. doi: 10.1128/jvi.76.23.11866-11879.2002.
Infected-cell protein 27 (ICP27) is an essential herpes simplex virus type 1 (HSV-1) regulatory protein that activates a subset of viral delayed-early and late genes, at least in part through posttranscriptional mechanisms. Previous studies have shown that the amino (N)-terminal half of the protein contains important functional regions, including a leucine-rich nuclear export signal (NES). However, to date, the phenotype of an HSV-1 ICP27 NES mutant has not been reported. In this study, we engineered and characterized dLeu, an HSV-1 deletion mutant that specifically lacks ICP27's NES (amino acids 6 to 19). The phenotype of dLeu was analyzed alongside those of eight other ICP27 N-terminal deletion mutants. We found that in Vero cells, dLeu displays modest defects in viral gene expression and an approximately 100-fold reduction in the production of viral progeny. Unlike wild-type (WT) ICP27, which exhibits a cytoplasmic distribution in addition to its predominant nuclear localization, dLeu ICP27 is highly restricted to the cell nucleus. This strongly suggests that the N-terminal leucine-rich sequence functions as an NES during viral infection. Our analysis of dLeu and the other mutants has enabled us to genetically define the regions in the N-terminal 200 residues of ICP27 which are required for efficient viral growth in Vero cells. Only two regions appear to be important: (i) the leucine-rich NES and (ii) the RGG box RNA-binding domain, encoded by residues 139 to 153. A virus lacking the RGG box-encoding sequence, d4-5, has a phenotype similar to that of dLeu in that it displays modest defects in viral gene expression and grows poorly. Interestingly, deletion of both the NES and RGG box, as well as the sequences in between, is lethal. The resulting virus, d1-5, displays severe defects in viral gene expression and DNA synthesis and is unable to produce significant amounts of infectious progeny. Therefore, the N-terminal portion of ICP27 contains at least two functional domains which collectively are absolutely essential for viral infection.
感染细胞蛋白27（ICP27）是1型单纯疱疹病毒（HSV-1）的一种必需调节蛋白，它至少部分地通过转录后机制激活一部分病毒延迟早期和晚期基因。先前的研究表明，该蛋白的氨基（N）末端一半包含重要的功能区域，包括富含亮氨酸的核输出信号（NES）。然而，迄今为止，尚未报道HSV-1 ICP27 NES突变体的表型。在本研究中，我们构建并鉴定了dLeu，这是一种HSV-1缺失突变体，它特异性地缺失了ICP27的NES（第6至19位氨基酸）。我们将dLeu的表型与其他八个ICP27 N末端缺失突变体的表型一起进行了分析。我们发现，在Vero细胞中，dLeu在病毒基因表达方面表现出适度的缺陷，病毒子代产量降低了约100倍。与野生型（WT）ICP27不同，WT ICP27除了主要定位于细胞核外还表现出细胞质分布，而dLeu ICP27高度局限于细胞核。这强烈表明，富含亮氨酸的N末端序列在病毒感染期间作为NES发挥作用。我们对dLeu和其他突变体的分析使我们能够从基因上确定ICP27 N末端200个残基中对于在Vero细胞中高效病毒生长所必需的区域。似乎只有两个区域很重要：（i）富含亮氨酸的NES和（ii）由第139至153位残基编码的RGG框RNA结合结构域。缺乏RGG框编码序列的病毒d4-5具有与dLeu相似的表型，即它在病毒基因表达方面表现出适度的缺陷且生长不良。有趣的是，NES和RGG框以及它们之间的序列同时缺失是致死的。产生的病毒d1-5在病毒基因表达和DNA合成方面表现出严重缺陷，并且无法产生大量有感染性的子代。因此，ICP27的N末端部分包含至少两个功能结构域，它们共同对于病毒感染绝对至关重要。
