Array analysis of viral gene transcription during lytic infection of cells in tissue culture with varicella-zoster virus
Array analysis of viral gene transcription during lytic infection of cells in tissue culture with varicella-zoster virus
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DOI：
10.1128/jvi.77.21.11718-11732.2003
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发表时间：
2003-11-01
影响因子：
5.4
通讯作者：
Gilden, DH
中科院分区：
文献类型：
--
作者：
Cohrs, RJ;Hurley, MP;Gilden, DH
水痘带状疱疹病毒（VZV）是一种嗜神经型甲疱疹病毒，可引起儿童水痘（水痘），潜伏于背根和自主神经节，并在几十年后重新激活，引起带状疱疹（带状疱疹）和其他神经系统并发症。虽然已经确定了VZV DNA的序列和结构，但对病毒基因在有效感染细胞中的表达知之甚少。这在一定程度上是因为VZV与细胞高度相关，并且没有产生用于同步感染的足够滴度的无细胞病毒。构建基于pcr的转录阵列，同时确定大约70个预测的VZV开放阅读帧（orf）的相对丰度。从每个ORF的5′和3′端扩增250 ~ 600 bp片段，并插入质粒载体。将病毒DNA插入物扩增、定量并在尼龙膜上标记。用VZV-感染细胞合成的放射性标记cDNA探测阵列显示，低多重性病毒感染后第1天至第3天，VZV基因的表达量增加，但相对丰度变化不大。最丰富的VZV转录本定位于ORF 9/ 9a、64、33/33A和49，其中只有ORF 9对应于先前鉴定的结构基因。阵列分析还将转录本定位到三个先前被认为是转录沉默的大基因间区域，随后通过Northern blot和逆转录pcr分析证实了这一结果。阵列分析为分析一种重要的普遍存在的人类病原体的转录提供了强大的工具。
Varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, causes childhood chickenpox (varicella), becomes latent in dorsal root and autonomic ganglia, and reactivates decades later to cause shingles (zoster) and other neurologic complications. Although the sequence and configuration of VZV DNA have been determined, relatively little is known about viral gene expression in productively infected cells. This is in part because VZV is highly cell associated, and sufficient titers of cell-free virus for use in synchronizing infection do not develop. PCR-based transcriptional arrays were constructed to simultaneously determine the relative abundance of the approximate to70 predicted VZV open reading frames (ORFs). Fragments (250 to 600 bp) from the 5' and 3' end of each ORF were PCR amplified and inserted into plasmid vectors. The virus DNA inserts were amplified, quantitated, and spotted onto nylon membranes. Probing the arrays with radiolabeled cDNA synthesized from VZV-infected cells revealed an increase in the magnitude of the expressed VZV genes from days 1 to 3 after low-multiplicity virus infection but little change in their relative abundance. The most abundant VZV transcripts mapped to ORFs 9/9A, 64, 33/33A, and 49, of which only ORF 9 corresponded to a previously identified structural gene. Array analysis also mapped transcripts to three large intergenic regions previously thought to be transcriptionally silent, results subsequently confirmed by Northern blot and reverse transcription-PCR analysis. Array analysis provides a formidable tool to analyze transcription of an important ubiquitous human pathogen.