DNA Microarrays for Neurotropic Human Herpesviruses

我们应用基于序列的计算机程序和我们自己的数据库，在HSV-1基因组中选择了75 nt的寡核苷酸序列，这些序列对单个病毒转录物的表达具有特异性。超过一半的病毒转录本可以用2- 3个短序列来唯一指定。我们将这些探针排列在化学活化的玻璃载玻片上，以产生“第一代”HSV-1 DNA微阵列（hsv芯片）。我们使用了镍翻译的病毒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特异性。我们还使用了oligo-dT引物cDNA，标记Cy3和cy5荧光核苷衍生物，这些衍生物是从感染HSV的细胞中分离出来的RNA，在各种条件下产生，这些条件会影响表达的基因类别。抑制新生蛋白合成只允许直接早期基因的表达，阻断DNA复制抑制严格晚期基因的表达，在感染后短时间内分离RNA导致早期转录物的高富集等。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检测出来。我们现在将：1。通过设计额外的病毒和细胞探针和标记方案来优化HSV-1 DNA微阵列，以提高特异性，特别是重叠病毒基因之间的区分。2. 利用芯片深入分析特定病毒基因、细胞类型、细胞分化状态对HSV基因表达全局规划的影响。这些实验将使用定义明确的重组病毒进行。在小鼠的生产和再激活感染期间测定病毒基因表达的全局模式。在这里，我们将充分利用特定病毒基因在病毒发病机制和潜伏期方面的作用。4. 将这些方法应用于人类α疱疹病毒组其他成员特异性DNA微阵列的设计。

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.