MOLECULAR BIOLOGY OF VARICELLA-ZOSTER VIRUS INFECTIONS

本实验室的主要基础研究重点涉及到 项目，目标如下： 1)鉴定、定位和鉴定水痘-带状疱疹病毒基因 以及潜伏感染或生产性感染中活跃的蛋白质。 2)确定基因表达的时间序列。 3)确定抗病毒药物与病毒基因的相互作用 产品通过耐药突变的分子分析。 为了实现这些目标，我们构建了各种 VZV全基因组重组文库。在.期间 在过去的一年里，我们已经完成了对更精细的映射和 病毒转录本的方向性。我们绘制了病毒IE 175 即刻早期基因。我们已经将该基因导入到 猴子细胞和稳定表达它的成熟细胞系。在……里面 表达该基因的细胞系似乎是 自动调节。使用我们可以用来测量 病毒基因启动子的活性我们检测到了一些VZV 反式激活和可反式激活的基因。在.期间 过去一年，我们还完成了对 10株VZV的胸苷激酶基因座，包括6株 由于阿昔洛韦基因的突变而产生抗药性。我们 确定了碱基突变的性质和位置 这种基因产物不起作用。分析中包括的是 首个从人身上发现的抗阿昔洛韦VZV毒株。 在过去的一年里，我们成功地建立了一个高度 灵敏、特异的35S原位杂交体系 标记的合成RNA探针。这种探针在VZV RNA中的表达 16名受试者中有6人的三叉神经节。持续的努力 将致力于更好地绘制和表征RNA 在VZV潜伏期表达。

The major basic research focus of this laboratory involves this project, with the following goals: 1) to identify, map and characterize varicella-zoster virus genes and proteins active in latent or productive infections. 2) to define the temporal sequence of gene expression. 3) to determine the interaction of antiviral drugs with viral gene products though a molecular analysis of drug-resistant mutants. To accomplish these ends we have constructed a variety of recombinant libraries of the complete VZV genome. During the past year we have concluded studies of the finer mapping and directionality of viral transcripts. We mapped the viral IE 175 immediate early gene. We have transfected that gene into monkey cells and developed cell lines which stably express it. In the cell lines the expression of the gene appears to be autoregulated. Using assays by which we could measure the activity of viral gene promotors we detected a number of VZV genes that are transactivating and transactivatible. During the past year we have also completed sequence analysis of the thymidine kinase locus of ten VZV strains, including six that are resistant to acyclovir by virtue of mutations in that gene. We identified the nature and location of base mutations which render this gene product nonfunctional. Included in the analyses is the first acyclovir-resistant VZV strain recovered from a human. During the past year we have successfully established a highly sensitive and specific in situ hybridization system using 35S labelled synthetic RNA probes. Such probes VZV RNA expression in the trigeminal ganglia of six of 16 subjects. Continuing efforts will be directed at better mapping and characterizing the RNA expressed during VZV latency.