HIV-MEDIATED REPRESSION OF MHC CLASS I GENE EXPRESSION

MHC I 类分子是病毒肽的主要受体 作为特定细胞毒性 T 淋巴细胞的靶标。 HIV-1感染 T 细胞系降低 I 类细胞表面表达和 使启动子活性降低高达 12 倍。镇压是 由 HIV-1 Tat 蛋白介导，源自剪接病毒 转录本，识别出这个双外显子 Tat 的新活性， 与 HIV LTR 的反式激活不同，两者共有 和两个外显子 Tat。 Tat 介导的镇压目标映射到 基础启动子。然而，Tat 不直接结合 DNA，也不 靶向 TATAA 或 Inr 基础启动子元件。相反，其 活性针对转录起始复合物。使用 酵母-二杂交筛选，我们鉴定了一个转录因子 与 Tat 相互作用，可能成为镇压的目标。研究 正在进一步表征这种相互作用及其 镇压中的作用。 Tat 蛋白功能域图谱 表明抑制和激活是由不同的介导的 和可分离的域。 Tat 阻遏蛋白活性取决于 C 末端 序列，而反式激活取决于 N 端序列；两者 函数需要核心序列。抑制活性需要 包含 Tat 第二外显子编码区域的结构域 基因，从氨基酸 73 开始，C 端限制在氨基酸之间 酸 80 和 83。Tat 阻遏物的功能也取决于其存在 位于蛋白质核心内的第 41 位赖氨酸。达 阻遏蛋白活性完全独立于两个 N 端结构域 对于反式激活、酸性片段和富含半胱氨酸至关重要 地区。相反，Tat 反式激活独立于第二个 Tat 的外显子编码区域。作为对这种新颖模型的进一步支持 可分离的 Tat 功能，我们已经证明在小鼠成纤维细胞中，Tat 抑制 I 类启动子活性，但不会反式激活 HIV 长期运输。我们认为不同的结构域介导了 Tat 的两个 功能不同的活动。

MHC class I molecules are the major receptors for viral peptides and serve as targets for specific cytotoxic T lymphocytes. HIV-1 infection of T cell lines decreases cell surface expression of class I and decreases the promoter activity by up to 12-fold. Repression is mediated by the HIV-1 Tat protein, derived from a spliced viral transcript, identifying a novel activity for this two-exon Tat, distinct from the transactivation of the HIV LTR common to both one- and two-exon Tat. The target of Tat-mediated repression maps to the basal promoter. However, Tat does not bind DNA directly, nor does it target either the TATAA or Inr basal promoter elements. Rather, its activity is directed to the transcription iniation complex. Using yeast-two hybrid screening, we have identified a transcription factor that interacts with Tat and may be the target for repression. Studies are in progress to further characterization this interaction, and its role in repression. Mapping of functional domains of Tat protein demonstrates that repression and activation are mediated by distinct and separable domains. Tat repressor activity depends on C-terminal sequences, whereas transactivation depends on N-terminal sequence; both functions require core sequences. The repressor activity requires a domain encompassing the region encoded by the second exon of the Tat gene, beginning at amino acid 73, with a C-terminal limit between amino acids 80 and 83. Tat repressor function also depends on the presence of a lysine at position 41, located within the core of the protein. Tat repressor activity is completely independent of two N-terminal domains essential for transactivation, the acidic segment and the cystein rich region. Conversely, Tat transactivation is independent of the second exon encoded region of Tat. As further support for this novel model of separable Tat functions, we have shown that in murine fibroblasts, Tat represses class I promoter activity, but does not transactivate the HIV LTR. We propose that distinct structural domains mediate Tat's two functionally distinct activities.