CHARACTERIZATION OF HUMAN CYTOMEGALOVIRUS-UL84 EARLY GENE AND IDENTIFICATION OF ITS PUTATIVE PROTEIN PRODUCT
CHARACTERIZATION OF HUMAN CYTOMEGALOVIRUS-UL84 EARLY GENE AND IDENTIFICATION OF ITS PUTATIVE PROTEIN PRODUCT
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DOI：
10.1128/jvi.66.2.1098-1108.1992
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发表时间：
1992-02-01
影响因子：
5.4
通讯作者：
HUANG, ES
中科院分区：
文献类型：
--
作者：
HE, YS;XU, L;HUANG, ES
分析了人巨细胞病毒早期基因UL84的DNA序列和转录模式。该基因被定位在人类巨细胞病毒基因组大独特片段0.534和0.545个图谱单元之间的2.6 kb PstI片段中，该片段与pp65和pp71基因相邻。与pp65和pp71基因的mRNA一样，一个2.0 kb的mRNA从该区域向左转录。该信息在感染后2.5 h首次被检测到，在感染后72 ~ 96 h达到最大水平。测定了2.6 kb PstI基因组DNA片段的核苷酸序列和该区域的cDNA。结果显示，一个位于cDNA多聚(a)尾部上游14个核苷酸的聚腺苷化信号（AATAAA）和一个1,761 bp的开放阅读框，能够编码65 kda的多肽。在114和135氨基酸之间的肽分子n端发现了一个潜在的亮氨酸拉链。此外，在氨基酸325和373之间发现了一个不同的亮氨酸周期重复序列，每8个位置有亮氨酸。通过引物延伸分析确定了该早期基因的转录起始位点。在帽位上游24bp处发现了一个推测的TATA盒（TATTTAA），在TATA盒上游的区域发现了几个反向重复序列。为了测试该cDNA的开放阅读框是否编码病毒特异性蛋白，我们在大肠杆菌中过表达该cDNA作为融合蛋白，用于兔体内产生抗体。在感染后6至72小时收获的感染细胞提取物中检测到分子大小为65 kDa的蛋白质，但在纯化的病毒粒子中未检测到。在病毒感染的晚期，细胞核和细胞质都有荧光。根据获得的结果，我们假设UL84可能是一种稳定的、病毒特异性的、能够形成同源或异二聚体分子的非结构蛋白。
The DNA sequence and transcription pattern of human cytomegalovirus early gene UL84 were analyzed. This gene was mapped within a 2.6-kb PstI fragment located between 0.534 and 0.545 map unit of the large unique segment of the human cytomegalovirus genome, which is adjacent to the pp65 and pp71 genes. A 2.0-kb mRNA was transcribed from this region in the same leftward direction as the mRNAs of the pp65 and pp71 genes. The message was first detected at 2.5 h postinfection and reached a maximal level between 72 and 96 h postinfection. The nucleotide sequences of the 2.6-kb PstI genomic DNA fragment and the cDNA derived from this region were determined. The resulting data revealed a polyadenylation signal (AATAAA) located 14 nucleotides upstream from the poly(A) tail of the cDNA and a 1,761-bp open reading frame capable of encoding a 65-kDa polypeptide. A potential leucine zipper was found in the N-terminal half of the peptide molecule between amino acids 114 and 135. In addition, a different periodic leucine repeat with leucine at every eighth position was found between amino acids 325 and 373. The transcriptional initiation site of this early gene was determined by primer extension analysis. A putative TATA box (TATTTAA) located 24 bp upstream of the cap site and several inverted repeats were found in the region further upstream of the TATA box. To test whether the open reading frame of this cDNA encodes a virus-specific protein, the cDNA was overexpressed in Escherichia coli as a fusion protein used to generate antibodies in rabbits. A protein with a molecular size of 65 kDa was detected in the infected-cell extracts harvested at 6 to 72 h postinfection, but not in purified virions, using immunoblot analysis. Both nuclear and cytoplasmic fluorescences were found at late stages of virus infection. From the results obtained, we postulate that UL84 may be a stable, virus-specific, nonstructural protein capable of forming a homo- or heterodimeric molecule.