HIV Tat-Mediated Repression of MHC Class I Expression

MHC-I类分子是病毒多肽的主要受体，是特异性细胞毒T淋巴细胞的靶标。感染HIV-1的T细胞株减少了细胞表面I类分子的表达，并使启动子活性降低了12倍。抑制是由HIV-1Tat蛋白介导的，该蛋白来自剪接的病毒转录本，鉴定了这个双外显子TAT的新活性，不同于单外显子和双外显子TAT共同的HIV LTR的反式激活。TAT抑制编码MHC I类重链和2-微球蛋白轻链的基因的转录。在这两种情况下，抑制都是通过核心启动子介导的。 TAT蛋白功能结构域的图谱表明，抑制和激活是由不同的和可分离的结构域介导的。TAT抑制物的活性依赖于C端序列，而反式激活依赖于N端序列；这两种功能都需要核心序列。抑制活性需要一个包含TAT基因第二外显子编码区域的区域，还依赖于位于蛋白质核心内的41位赖氨酸的存在。TAT抑制物的活性完全不依赖于反式激活所必需的两个N末端结构域，即酸性部分和富含半胱氨酸的区域。相反，TAT反式激活不依赖于TAT的第二外显子编码区。为了进一步支持这一新的可分离TAT功能模型，我们已经证明在小鼠成纤维细胞中，TAT抑制I类启动子的活性，但不反式激活HIV LTR。我们认为，不同的结构域介导了TAT的两个不同的功能活动。 通过酵母双杂交筛选，我们已经确定TAT与通用转录因子TFIID的TAFII250成分相互作用。TAT与TAFII250的相互作用是广泛的。TAT的C-末端结构域是抑制所必需的，它与TAFII250的一个包含其HAT结构域的片段结合。TAT的N-末端片段包含其激活结构域，与位于885-984aa和1120-1297aa之间的TAFII250的两个不连续片段结合。TAT与TAFII250的885-984aa片段的结合需要TAT的半胱氨酸富集区，而不是酸性或谷氨酰胺富集区。TAT的N-末端结构域与1120-1279aa片段的结合不涉及酸性、半胱氨酸或谷氨酰胺富集区。有趣的是，我们已经证明HIV LTR的转录不依赖于TAFII250，这可能是其抵抗TAT介导的抑制的原因。

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.