BIOLOGICAL VARIATION OF EQUINE INFECTIOUS ANEMIA VIRUS

描述：（改编自申请人的摘要） 这个项目是为了了解控制 慢病毒复制和体内表达。 具体目标是 在功能上表征EIAV的LTR和rev基因的可变区。 LTR变异的生物学意义将在 使用CAT（氯霉素乙酰转移酶）的瞬时表达测定 作为报告基因。 为了确定LTR的可变性是否在以下方面发挥作用： 细胞嗜性，研究者建议在细胞中进行测定 先前显示出在体内和体内的容许性不同， EIAV的体外分离株。 这些研究的结果可能会揭示， 细胞转录因子在病毒基因调控中发挥作用 表情 如果是这样的话，凝胶阻滞试验和DNA足迹法将是可行的。 用于确认细胞DNA结合蛋白的存在， 鉴定细胞特异性调节病毒所必需LTR序列 表情 EIAV S3开放阅读框架的变异性表明， Rev感受态和Rev缺陷型基因型在体内共存。 到 确定这是否为真，申请人建议亚克隆S3变体 转化为EIAV的全长前病毒克隆。 将分析Rev函数 通过定量细胞核中差异剪接的mRNA转录物， 转染细胞的细胞质部分。 转速变化对 病毒蛋白的表达将使用 放射免疫沉淀 研究人员预计，这些研究 可能表明Rev缺陷型基因型在体内 限制病毒基因表达和维持持久的 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.