HIV Tat-Mediated Repression of MHC Class I Expression

MHC I类分子是病毒肽的主要受体，并作为特异性细胞毒性T淋巴细胞的靶标。HIV-1感染的T细胞系降低细胞表面表达的I类和降低启动子活性高达12倍。抑制由HIV-1达特蛋白介导，该蛋白来源于剪接的病毒转录物，鉴定了该双外显子达特的新活性，不同于单外显子和双外显子达特共有的HIV LTR的反式激活。达特抑制编码MHC I类重链和β 2-微球蛋白轻链的基因的转录。在这两种情况下，阻遏都是通过核心启动子介导的。 达特蛋白功能域的定位表明，阻遏和激活是由不同的和可分离的结构域介导的。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;两种功能都需要核心序列。阻遏物活性需要包含由达特基因的第二外显子编码的区域的结构域，并且还取决于位于蛋白质核心内的位置41处赖氨酸的存在。达特阻遏物的活性是完全独立的两个N-末端结构域的反式激活所必需的，酸性段和半胱氨酸丰富的区域。相反，达特反式激活不依赖于达特的第二外显子编码区。作为对这种分离达特功能的新模型的进一步支持，我们已经表明，在鼠成纤维细胞中，达特抑制I类启动子活性，但不反式激活HIV LTR。我们认为不同的结构域介导了达特的两种功能不同的活动。 使用酵母双杂交筛选，我们已经确定，达特与一般的转录因子，TFIID的TAFII 250组件相互作用。达特和TAFII 250之间的相互作用是广泛的。达特的C-末端结构域是阻遏所必需的，其结合到包含其HAT结构域的TAFII 250的区段。达特的N-末端片段包含其活化结构域，其与位于885- 984 aa和1120- 1297 aa之间的TAFII 250的两个不连续片段结合。达特与TAFII 250的885- 984 aa区段的结合需要达特的富含半胱氨酸的结构域，但不需要酸性或富含谷氨酰胺的结构域。达特的N-末端结构域与1120- 1279 aa区段的结合不涉及酸性、半胱氨酸或谷氨酰胺富集结构域。有趣的是，我们已经证明HIV LTR的转录不依赖于TAFII 250，这可能解释了其对达特介导的抑制的抗性。

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. Tat represses transcription of both the genes encoding the MHC class I heavy chain and the 2-microglobulin light chain. In both cases, repression is mediated through the core promoter. 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 and 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. Using yeast-two hybrid screening, we have determined that Tat interacts with the TAFII250 component of the general transcription factor, TFIID. The interaction between Tat and TAFII250 is extensive. The C-terminal domain of Tat, which is necessary for repression, binds to a segment of TAFII250 that encompasses its HAT domain. The N-terminal segment of Tat, which contains its activation domains binds to two discontinuous segments of TAFII250 located between 885-984aa and 1120-1297aa. Binding of Tat to the 885-984aa segment of TAFII250 requires the cystein rich domain of Tat, but not the acidic or glutamine rich domains. Binding by the N-terminal domain of Tat to the 1120-1279aa segment does not involve the acidic, cysteine or glutamine-rich domains. Interestingly, we have demonstrated that transcription of the HIV LTR does not depend on TAFII250 which may account for its resistance to Tat mediated repression.