BIOLOGICAL VARIATION OF EQUINE INFECTIOUS ANEMIA VIRUS

描述：(改编自申请者摘要)长期目标 这个项目是为了了解控制 慢病毒在体内复制和表达。具体目标是 EIAV LTRV和REV基因可变区的功能特征 LTR变异的生物学意义将在#年进行研究 用CAT(氯霉素乙酰转移酶)检测瞬时表达 作为一名记者基因。为了确定LTR中的可变性是否在 细胞嗜性，研究人员建议在细胞中进行分析 以前显示的在体内的容许性不同--和在 EIAV的体外分离株。这些研究的结果可能会揭示出 细胞转录因子在病毒基因调控中的作用 表情。如果是这样的话，凝胶延迟分析和DNA足迹将 用于确认细胞DNA结合蛋白的存在，并 确定病毒细胞特异性调控所需的LTR序列 表情。EIAV S3开放阅读框架的可变性表明 活体内同时存在REV活性和REV缺陷两种基因型。至 确定这是否属实，申请人提议亚克隆S3变体 变成了EIAV的全长前病毒克隆。将分析REV函数 通过对核和核中差异剪接的mRNA转录本进行定量 转基因细胞的胞质组分。转速变化对车速的影响 病毒蛋白的表达将通过以下方法确定 放射免疫沉淀。调查人员预计，这些研究 可能表明rev缺陷基因在体内起着重要的作用。 病毒基因表达的限制与持续性的维持 感染。

DESCRIPTION: (Adapted from applicant's abstract) The long range goals of this project are to understand molecular mechanisms which control lentivirus replication and expression in vivo. The specific aims are to functionally characterize variable regions in the LTR and rev gene of EIAV. The biological significance of variation in the LTR will be studied in transient expression assays using CAT (chloramphenicol acetyl transferase) as a reporter gene. To determine if variability in the LTR plays a role in cell tropism, the investigator proposes performing assays in cells previously shown to differ in permissiveness for in vivo- and in vitro-derived isolates of EIAV. Results of these studies may reveal if cellular transcription factors play a role in regulation of viral gene expression. If so, gel retardation assays and DNA footprinting will be used to confirm the presence of cellular DNA binding proteins and to identify LTR sequences necessary for cell-specific regulation of viral expression. Variability in the S3 open reading frame of EIAV suggests that both rev-competent and rev-defective genotypes co-exist in vivo. To determine if this is true, the applicant proposes to subclone S3 variants into a full-length proviral clone of EIAV. Rev function will be analyzed by quantitating differentially spliced mRNA transcripts in nuclear and cytoplasmic fractions of transfected cells. The effect of rev variation on expression of viral proteins will be determined using radioimmunoprecipitation. The investigator anticipates that these studies may indicate that rev-defective genotypes play an important role in vivo in restriction of viral gene expression and maintenance of persistent infection.