REGULATION OF SIMIAN FOAMY VIRUS GENE EXPRESSION

猿泡沫病毒基因表达的调控

• 批准号: 2065672
• 负责人: Paul Luciw
• 金额: $ 18.04万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2065672
• 关键词: CHO cells; Retroviridae; antibody; gene expression; genetic manipulation; genetic regulation; molecular cloning; nucleic acid repetitive sequence; plasmids; posttranscriptional RNA processing; transcription factor; virion; virus protein

The foamy viruses, or syncytium-forming viruses, are members of the spumaviriniae sub-family of retroviruses. Foamy viruses are found in many mammalian species and appear to be non-pathogenic in their natural hosts even though they have a wide tissue range and induce extensive cytopathology in cell cultures. The genomes of simian foamy virus type 1 (SFV-l) from rhesus macaque and human spumaretrovirus (HSRV) have been molecularly cloned and sequenced; pairwise comparisons of these two viruses show from 30% to 80% homology, depending on the region selected for analysis. Both viruses encode several open reading frames (ORFs) in addition to genes for virion structural proteins. Recent studies have revealed that SFV-1 and HSRV each encode a transactivator (taf) which functions to strongly augment transcription directed by the viral long terminal repeat (LTR). The main objective of this proposal is to elucidate molecular mechanisms regulating SFV-1 gene expression directed by the viral LTR. It remains to be determined whether taf acts directly on the LTR or whether transactivation is mediated by cellular factors. The target (TAR) for taf is distributed over two parts of the U3 domain of the viral LTR upstream from the TATA box in the viral promoter. Each these two TAR regions is about 300 base pairs (bp) long and no homologies (i.e., repeat elements) are detected between the two TARs. In addition, the promoter-distal TAR (TAR-d) functions in only in the sense orientation whereas the promoter-proximal TAR (TAR-p) functions in either orientation. These observations (on the complex nature of the targets) support the hypothesis that taf may function through more than one cellular factor which interacts with different sequence elements in the U3 region of the LTR. SPECIFIC AIM 1: Binding sites for cellular factors and the precise target sequences for taf in the SFV-1 LTR will be defined. SPECIFIC AIM 2: Monospecific antibodies will be made to detect the SFV-1 taf gene product; taf protein in infected cells will be extensively characterized. SPECIFIC AIM 3: Genetically engineered yeast and mammalian cells will be used to produce the SFV-1 taf gene product for structural and functional studies. SPECIFIC AIM 4: To elucidate the mechanism of SFV-1 transcriptional transactivation, functional domains of taf will be identified by mutagenesis and by evaluation of hybrid transactivators. These studies on SFV-1 regulation are relevant for elucidating mechanisms which control viral latency (or persistent infection) in the host animal. Observations made in the course of the proposed research will also enhance the understanding of mechanisms controlling eucaryotic transcription; the proposed studies on SFV-1 gene expression are significant since new (cellular) transcriptional factors may be identified.

泡沫病毒或合胞体形成病毒是 逆转录病毒的马病毒亚科。 泡沫病毒存在于 许多哺乳动物物种，似乎是非致病性的，在他们的自然 尽管它们具有广泛的组织范围并诱导广泛的 细胞培养物中的细胞病理学。 猴泡沫病毒1型的基因组 来自恒河猴的SFV-1和人疱疹病毒（HSRV）已经被研究。 分子克隆和测序;这两个配对比较 病毒显示30%至80%的同源性，这取决于所选择的区域 用于分析 两种病毒都编码几个开放阅读框（ORF）， 此外还有病毒体结构蛋白基因。 最近的研究 揭示了SFV-1和HSRV各自编码一个反式激活因子（taf）， 其功能是强烈增强病毒长链指导的转录 末端重复序列（LTR）。 这项建议的主要目的是 阐明调控SFV-1基因表达的分子机制 被病毒LTR感染 TAF是否直接采取行动还有待确定 或反式激活是否由细胞因子介导。 taf的目标（TAR）分布在U3域的两个部分上 病毒启动子中TATA盒上游的病毒LTR。 每个 这两个TAR区域长约300个碱基对（bp），并且没有同源性 (i.e.,重复元件）在两个TAR之间被检测到。 此外，本发明还提供了一种方法， 启动子远端TAR（TAR-d）仅在以下意义上起作用： 而启动子近端TAR（TAR-p）在 导向 这些意见（关于目标的复杂性） 支持TAF可能通过多个机制发挥作用的假设。 细胞因子，与不同的序列元件相互作用， LTR的U3区。 特定目的1：细胞因子的结合位点和精确靶点 将定义SFV-1 LTR中的taf序列。 特异性目的2：制备单特异性抗体以检测SFV-1 taf基因产物;感染细胞中的taf蛋白将被广泛地 表征了 具体目标3：基因工程酵母和哺乳动物细胞将被 用于产生SFV-1 taf基因产物，用于结构和功能 问题研究 具体目的4：阐明SFV-1转录的机制 反式激活，TAF的功能结构域将通过 诱变和评价杂合反式激活因子。 这些关于SFV-1调节的研究与阐明机制有关 其控制宿主动物中的病毒潜伏期（或持续感染）。 在拟议的研究过程中所作的观察也将 增强对真核生物控制机制的理解 转录; SFV-1基因表达的拟议研究是 因为新的（细胞）转录因子可能 鉴定