Hibbard M K, Sandri-Goldin R M
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717-4025, USA.
J Virol. 1995 Aug;69(8):4656-67. doi: 10.1128/JVI.69.8.4656-4667.1995.
The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP27 is an essential regulatory protein that localizes to the nuclei of infected cells. The strong nuclear localization signal (NLS) of ICP27 was identified recently and shown to reside in the amino-terminal portion of the polypeptide from residues 110 to 137 (W.E. Mears, V. Lam, and S.A. Rice, J. Virol. 69:935-947, 1995). There are also two arginine-rich regions directly succeeding the NLS. The first of these arginine-rich sequences (residues 141 to 151), together with the NLS, has been shown by Mears et al. to form the nucleolar localization signal. Arginine-rich motifs are common in domains involved in nuclear localization and RNA binding. To analyze the role of the arginine-rich regions in ICP27, we constructed stably transformed cell lines containing ICP27 mutants with deletions of all or parts of the NLS and arginine-rich regions. We also constructed mutants in which these regions were replaced with heterologous NLSs or RNA-binding domains. Characterization of these mutants indicated that the arginine-rich regions were required but not sufficient for wild-type localization of ICP27. More importantly, the NLS and arginine-rich regions were also essential to the function of ICP27. Mutants lacking these sequences were defective in late gene expression during infection even when ICP27 was properly localized to the nucleus by substitution of the NLS from simian virus 40 large T antigen. Further, the defect in late gene expression could not be overcome by replacement with the highly basic RNA-binding domain of human immunodeficiency virus type 1 Tat. The deficiency in late gene expression was independent of ICP27's role in stimulating viral DNA replication. In addition, localization of the HSV-1 proteins ICP4, ICP0, and ICP8 was unaffected by ICP27 mutants in this region. These results suggest that the arginine-rich regions are required for efficient nuclear localization and for the regulatory activity of ICP27 involved in viral late gene expression.
单纯疱疹病毒1型（HSV - 1）的立即早期蛋白ICP27是一种必需的调节蛋白，定位于受感染细胞的细胞核中。ICP27强大的核定位信号（NLS）最近已被确定，且显示位于多肽的氨基末端部分，从第110位氨基酸残基至第137位氨基酸残基（W.E. Mears、V. Lam和S.A. Rice，《病毒学杂志》69:935 - 947，1995年）。在NLS之后还有两个富含精氨酸的区域。Mears等人已证明，这些富含精氨酸序列中的第一个（第141位氨基酸残基至第151位氨基酸残基）与NLS一起构成核仁定位信号。富含精氨酸的基序在参与核定位和RNA结合的结构域中很常见。为了分析ICP27中富含精氨酸区域的作用，我们构建了稳定转化的细胞系，这些细胞系含有缺失全部或部分NLS和富含精氨酸区域的ICP27突变体。我们还构建了将这些区域替换为异源NLS或RNA结合结构域的突变体。对这些突变体的表征表明，富含精氨酸的区域对于ICP27的野生型定位是必需的，但并不充分。更重要的是，NLS和富含精氨酸的区域对于ICP27的功能也是必不可少的。即使通过替换来自猿猴病毒40大T抗原的NLS使ICP27正确定位于细胞核，缺乏这些序列的突变体在感染期间的晚期基因表达仍存在缺陷。此外，用人类免疫缺陷病毒1型Tat的高度碱性RNA结合结构域进行替换并不能克服晚期基因表达的缺陷。晚期基因表达的缺陷与ICP27在刺激病毒DNA复制中的作用无关。此外，HSV - 1蛋白ICP4、ICP0和ICP8的定位不受该区域ICP27突变体的影响。这些结果表明，富含精氨酸的区域对于有效的核定位以及ICP27参与病毒晚期基因表达的调节活性是必需的。
