IDENTIFICATION OF THE LYTIC ORIGIN OF DNA-REPLICATION IN HUMAN CYTOMEGALOVIRUS BY A NOVEL-APPROACH UTILIZING GANCICLOVIR-INDUCED CHAIN TERMINATION
IDENTIFICATION OF THE LYTIC ORIGIN OF DNA-REPLICATION IN HUMAN CYTOMEGALOVIRUS BY A NOVEL-APPROACH UTILIZING GANCICLOVIR-INDUCED CHAIN TERMINATION
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DOI：
10.1128/jvi.64.12.6184-6195.1990
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发表时间：
1990-12-01
影响因子：
5.4
通讯作者：
HAYWARD, GS
中科院分区：
文献类型：
--
作者：
HAMZEH, FM;LIETMAN, PS;HAYWARD, GS
在抗病毒核苷酸类似物更昔洛韦存在的情况下，感染人类巨细胞病毒会导致持续低水平的病毒DNA合成，并积累相对较小的双链后代DNA片段。这些片段一直被证明是对位于唯一的L片段中心附近的一小段病毒基因组(EcoRI-V)序列的扩增。进一步的图谱显示，这些片段中所代表的病毒序列是以丰度梯度出现的，从接近0.35到0.4个地图单位的点在两个方向上递减。扩增序列所占比例随感染时间和更昔洛韦用量的增加而增加。我们的结论是，人巨细胞病毒的主要裂解循环复制起点位于单链DNA结合蛋白(DB140)启动子的上游和右侧的3-4kb区域。扩增的含有来源的DNA分子似乎是通过对基因组的截断片段进行连续几轮的双向启动而产生的，这些截断片段是由于将更昔洛韦掺入病毒DNA而产生的链终止效应的结果。对Ori-Lyt附近的DNA序列的检查发现了一个很大的复杂的上游区域，它可能是一个非编码的基因间隔区，与之前描述的任何疱疹病毒起源没有同源性。这个2.5kb的区域包括许多重复和反向序列，以及共同的Cre/ATF和其他转录因子结合位点，以及一组有趣的23个副本的散布的Decamer共同元件AAAACACCGT，该元件在猿猴巨细胞病毒中也是保守的。这项工作代表了首次鉴定巨细胞病毒基因组中的起始域，也是首次证明了疱疹病毒裂解周期起源的双向机制。
Infection with human cytomegalovirus in the presence of the antiviral nucleotide analog ganciclovir results in continuing low-level viral DNA synthesis and the accumulation of relatively small fragments of double-stranded progeny DNA. These fragments consistently proved to represent amplification of sequences from only one small section of the viral genome (EcoRI-V) lying near the center of the unique L segment. Further mapping revealed that the viral sequences represented in these fragments occurred in gradients of abundance that decreased in both directions from a point near 0.35 to 0.4 map unit. The proportion of amplified sequences increased with both time after infection and dosage of ganciclovir used. We conclude that the primary lytic cycle replication origin of human cytomegalovirus lies within a 3- to 4-kb region immediately upstream and to the right of the promoter for the single-stranded DNA-binding protein (DB140). The amplified origin-containing DNA molecules appeared to arise by continuing rounds of bidirectional initiation on truncated fragments of the genome that were generated as a result of chain termination effects induced by the incorporation of ganciclovir into the viral DNA. Inspection of the DNA sequence in the vicinity of ori-Lyt revealed a large complex upstream region that may be a noncoding intergenic domain and that bears no homology to any previously described herpesvirus origin. This 2.5-kb region includes many duplicated and inverted sequences, together with consensus CRE/ATF and other transcription factor-binding sites, and an interesting set of 23 copies of an interspersed decamer consensus element AAAACACCGT that is also conserved at the equivalent locus in simian cytomegalovirus. This work represents the first identification of an origin domain in a cytomegalovirus genome and is the first demonstration of a bidirectional mechanism for any herpesvirus lytic cycle origin.