Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6
Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6
复制标题
鉴定映射到人类疱疹病毒 6 推定的早期基因座的反式激活功能
作者：
Michelle E. D. Martin;John Nicholas;B. Thomson;C. Newman;R. Honess
测序研究表明，人类疱疹病毒6型（HHV-6）基因组的独特组成部分和人类巨细胞病毒基因组的独特长片段在遗传上是共线的。特别令人感兴趣的是HHV-6基因组中局部CpG二核苷酸抑制区域的鉴定，这是人巨细胞病毒、鼠巨细胞病毒和猿巨细胞病毒基因组中保守的特征，也是这些病毒主要立即早期基因座的特征。 HHV-6 中与该区域相邻的是大约 30 个 103 至 108 bp 序列元件的拷贝，其中除了单个 KpnI 识别位点外，还包含转录因子 AP2 和 NF kappa B 的共有结合位点。这些 KpnI 重复单元一起可以组成立即早期增强子，类似于巨细胞病毒中发现的增强子。我们展示了 HHV-6 该区域的序列，并证明该区域编码反式激活功能。我们使用聚合酶链式反应来合成包含开放阅读框和带有或不带有上游 KpnI 重复单元的 5' 序列的片段。在 Vero 细胞和外周血淋巴细胞的共转染实验中，含有这些 HHV-6 编码和 5' 序列的效应质粒能够激活异源启动子-氯霉素乙酰转移酶 (CAT) 构建体，包括腺病毒 E3-CAT 和 E4-CAT、人 T 细胞嗜淋巴细胞病毒 I 型长末端重复序列 (LTR)-CAT 和人免疫缺陷病毒 LTR-CAT。此外，我们已经确定了主要开放阅读框（RF4；2.3 kb）对于激活至关重要，并且我们已经证明，人类免疫缺陷病毒 LTR 中的 NF kappa B、SP1 和 TATA 盒基序都是 HHV-6 编码的反式激活子完全诱导启动子所必需的。
Sequencing studies have indicated that the unique component of the human herpesvirus 6 (HHV-6) genome and the unique long segment of the human cytomegalovirus genome are genetically colinear. Of particular interest is the identification of a region of local CpG dinucleotide suppression in the genome of HHV-6, a feature conserved in the genomes of human cytomegalovirus, murine cytomegalovirus, and simian cytomegalovirus, and a characteristic of the major immediate-early loci of these viruses. Adjacent to this region in HHV-6 are approximately 30 copies of a 103- to 108-bp sequence element, which contains consensus binding sites for the transcription factors AP2 and NF kappa B, in addition to a single KpnI recognition site. Together, these KpnI repeat units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We present the sequence of this region of HHV-6 and demonstrate that a transactivating function is encoded by this region. We have used polymerase chain reaction to synthesize fragments containing open reading frames and 5' sequences with or without the upstream KpnI repeat units. Effector plasmids containing these HHV-6 coding and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase (CAT) constructs, including adenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus type I long terminal repeat (LTR)-CAT, and human immunodeficiency virus LTR-CAT, in cotransfection experiments in Vero cells and peripheral blood lymphocytes. Furthermore, we have identified the major open reading frame (RF4; 2.3 kb) as being essential for activation, and we have shown that the NF kappa B, SP1, and TATA box motifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the HHV-6-encoded transactivator.