HIV-MEDIATED REPRESSION OF MHC CLASS I GENE EXPRESSION

MHC I类分子是病毒肽的主要受体， 作为特异性细胞毒性T淋巴细胞的靶点。hiv-1感染 T细胞系的表达降低了I类细胞的细胞表面表达， 启动子活性降低了12倍。抑制是介导的 通过HIV-1达特蛋白，从一个剪接的病毒转录物衍生， 鉴定这种两外显子达特的新活性，其不同于 单外显子和双外显子达特共有的HIV LTR的反式激活。的 Tat介导的阻遏的靶点映射到基础启动子。然而，在这方面， 达特不直接结合DNA，也不靶向TATAA或 inr基础启动子元件。相反，它的活动是针对 转录起始复合体使用酵母双杂交筛选，我们 已经鉴定了一种与达特相互作用的转录因子， 成为镇压的目标。研究正在进行中， 描述这种相互作用及其在抑制中的作用。在in 初步的体外分析，我们已经验证了相互作用的 带有达特的TFIID组件。此外，在体外转录的MHC I类启动子被达特抑制。 达特蛋白功能结构域的定位表明， 抑制和激活是由不同的和可分离的 域.达特阻遏物活性依赖于C-末端序列，而 反式激活依赖于N-末端序列;这两种功能都需要 核心序列阻遏物的活性需要一个包含 由达特基因的第二外显子编码的区域，开始于 氨基酸73，C-末端限制在氨基酸80和83之间。 达特阻遏物的功能也依赖于在细胞内赖氨酸的存在。 位置41，位于蛋白质的核心内。达特阻遏物 活性完全独立于两个N-末端结构域 对于反式激活，酸性片段和富含半胱氨酸的区域。 相反，达特反式激活不依赖于第二外显子 达特的编码区。作为对这一新模式的进一步支持， 分离的达特功能，我们已经表明，在鼠成纤维细胞，达特， 抑制I类启动子活性，但不反式激活HIV LTR我们提出不同的结构域介导达特的两个 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 characterize this interaction, and its role in repression. In in preliminary in vitro analysis, we have verified the interaction of the TFIID component with Tat. Furthermore, in vitro transcription of the MHC class I promoter is repressed by Tat. 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.