Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2.
Conserved regions in the Epstein-Barr virus leader protein define distinct domains required for nuclear localization and transcriptional cooperation with EBNA2.
复制标题
Epstein-Barr 病毒前导蛋白中的保守区域定义了核定位和与 EBNA2 转录合作所需的不同结构域。
DOI：
10.1128/jvi.74.21.9953-9963.2000
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发表时间：
2000
影响因子：
5.4
通讯作者：
Ling,PD
中科院分区：
文献类型：
--
作者：
Peng,R;Tan,J;Ling,PD
EB病毒（EBV）EBNA-LP是一种潜伏蛋白，其功能尚未完全了解。最近的研究表明，EBNA-LP可能是一个重要的EBNA 2辅因子，通过增强EBNA 2刺激潜伏C和LMP-1启动子。为了进一步了解EBNA-LP功能，我们将一系列突变引入进化上保守的区域，并测试突变蛋白增强EBNA 2刺激潜伏C和LMP-1启动子的能力。三个保守区域（CR 1至CR 3）位于EBNA 2协同功能所必需的重复结构域中。此外，三个丝氨酸残基在重复结构域中也很保守。在CR 1至CR 3中引入了保守的丙氨酸突变，并且在具有两个重复的EBNA-LP中保守的丝氨酸也被改变为丙氨酸残基，这是能够与EBNA 2合作的最小蛋白质。引入CR 1a的突变对EBNA-LP功能没有影响，而引入CR 1b的突变导致EBNA-LP活性略有下降。CR 1c和CR2的突变导致蛋白质不再只定位于细胞核，也没有EBNA 2合作活性。引入保守丝氨酸S5/71中的突变导致具有略微更高活性的蛋白质，而引入保守丝氨酸S35/101或CR 3（其含有S60/126）中的突变导致具有显著降低的活性的EBNA-LP蛋白质。还通过将编码这些带正电荷的氨基酸组的寡核苷酸引入细胞质测试蛋白（单纯疱疹病毒Δ IE 175）中，并通过检查所得蛋白的细胞内定位，检查了EBNA-LP CR 1c和CR2中的潜在亲核信号。该测定鉴定了包含CR 2的EBNA-LP氨基酸43和50（第二个W重复中的109至117）之间的强核定位信号，而EBNA-LP氨基酸29至36（第二个W重复中的91至98）无法独立发挥核定位信号的作用。然而，氨基酸29至50的组合导致比单独氨基酸43至50更有效的核定位。这些结果表明，EBNA-LP具有二分核定位信号，有效的核定位是EBNA 2协同功能所必需的。有趣的是，EBNA-LP只有一个单一的重复定位专门的细胞质，提供了一个解释，为什么这种亚型没有活动。此外，两个保守的丝氨酸残基，这是不同的核输入功能是重要的EBNA 2协同功能。
Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus ΔIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.