DNA Microarrays for Neurotropic Human Herpesviruses

用于嗜神经人类疱疹病毒的 DNA 微阵列

• 批准号: 6756450
• 负责人: Timothy F Osborne
• 金额: $ 30.92万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6756450
• 关键词: gene expression; herpes simplex virus 1; herpes simplex virus 2; host organism interaction; laboratory mouse; microarray technology; neurotropic virus; nucleic acid probes; oligonucleotides; recombinant virus; regulatory gene; varicella zoster virus; virus DNA; virus genetics; virus infection mechanism

We have applied sequence-based computer programs and our own databases to select 75 nt oligonucleotide sequences within the HSV-1 genome that are specific for the expression of individual viral transcripts. Better than half of the total viral transcripts can be uniquely specified with sets of 2--3 short sequences. We have arrayed these probes on chemically activated glass slides to generate a "first generation" HSV-1 DNA microarray (HSV-chip). We have used nick-translated viral DNA and cloned DNA fragments to optimize hybridization conditions and demonstrate the high specificity of this first generation chip. We have also used oligo-dT primed cDNA labeled with Cy3 and Cy5-luorescent nucleoside derivatives generated from RNA isolated from cells infected with HSV under various conditions, which influence the class of genes expressed. Inhibition of de novo protein synthesis allows only expression of immediate-early genes, blockage of DNA replication inhibits expression of strict late genes, isolation of RNA at short times after infection results in high enrichment of early phase transcripts, etc. Our preliminary results confirm that all classes of viral transcripts can be detected with good efficiency and very high specificity. We will now: 1. Optimize an HSV-1 DNA microarray by designing additional viral and cellular probes and labeling regimens to increase specificity and especially the discrimination between overlapping viral genes. 2. Apply the chips to the in-depth analysis of the influence of specific viral genes, cell types, and state of cell differentiation on the global program of HSV gene expression. These experiments will be carried out using well- defined recombinant viruses.3. Assay global patterns of viral gene expression during productive and reactivating infection in the mouse. Here we will take advantage of the well documented roles of specific viral genes in aspects of viral pathogenesis and latency. 4. Apply these methods towards design of DNA microarrays specific for other members of the human alpha herpesvirus group.

我们应用基于序列的计算机程序和我们自己的数据库来选择HSV-1基因组中针对单个病毒转录本表达的75个核苷酸寡核苷酸序列。超过一半的病毒转录本可以用2-3个短序列的集合唯一地指定。我们将这些探针排列在化学激活的玻片上，生成了第一代HSV-1 DNA微阵列(HSV芯片)。我们利用缺口翻译的病毒DNA和克隆的DNA片段来优化杂交条件，并证明了这种第一代芯片的高度特异性。我们还使用了在不同条件下从HSV感染细胞中分离的RNA产生的Cy3和Cy5荧光核苷衍生物标记的寡聚脱氧核糖核酸，它们影响了基因表达的类别。抑制从头蛋白的合成只允许即刻早期基因的表达，阻断DNA复制可以抑制严格的晚期基因的表达，感染后短时间分离RNA会导致早期转录本的高度浓缩等。我们的初步结果证实，所有类型的病毒转录本都能以良好的效率和非常高的特异性被检测出来。我们现在将：1.通过设计额外的病毒和细胞探针以及标记方案来优化HSV-1DNA微阵列，以提高特异性，特别是重叠病毒基因之间的区分性。2.应用这些芯片深入分析特定的病毒基因、细胞类型和细胞分化状态对HSV基因表达的全局程序的影响。这些实验将使用定义明确的重组病毒进行。分析病毒基因在小鼠产生感染和重新激活感染期间的全球表达模式。在这里，我们将充分利用特定病毒基因在病毒致病机制和潜伏期方面所发挥的作用。4.将这些方法应用于针对人类阿尔法疱疹病毒组其他成员的DNA微阵列的设计。

项目成果

Herpes simplex virus type 1 shows multiple interactions with sulfonated compounds at binding, penetration, and cell-to-cell passage.

1 型单纯疱疹病毒在结合、渗透和细胞间传代时与磺化化合物表现出多种相互作用。

• DOI: 10.1007/s11262-006-0016-5
• 发表时间: 2007
• 期刊: Virus genes
• 影响因子: 1.6
• 作者: Aguilar,JoséSantiago;Held,KatherineS;Wagner,EdwardK
• 通讯作者: Wagner,EdwardK

Design of a herpes simplex virus type 2 long oligonucleotide-based microarray: global analysis of HSV-2 transcript abundance during productive infection.

• DOI: 10.1385/1-59259-848-x:423
• 发表时间: 2005
• 期刊: Methods in molecular biology
• 作者: J. S. Aguilar;P. Ghazal;E. Wagner

The TATGARAT box of the HSV-1 ICP27 gene is essential for immediate early expression but not critical for efficient replication in vitro or in vivo.

HSV-1 ICP27 基因的 TATGARAT 盒对于立即早期表达至关重要，但对于体外或体内的有效复制并不重要。

• DOI: 10.1007/s11262-004-7437-9
• 发表时间: 2004
• 期刊: Virus genes
• 影响因子: 1.6
• 作者: Sun,Aixu;Devi-Rao,GV;Rice,MK;Gary,LW;Bloom,DC;Sandri-Goldin,RM;Wagner,P;Wager,EK
• 通讯作者: Wager,EK