MULTIPLE FUNCTIONAL DOMAINS OF TAT, THE TRANS-ACTIVATOR OF HIV-1, DEFINED BY MUTATIONAL ANALYSIS
MULTIPLE FUNCTIONAL DOMAINS OF TAT, THE TRANS-ACTIVATOR OF HIV-1, DEFINED BY MUTATIONAL ANALYSIS
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DOI：
10.1093/nar/17.9.3551
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发表时间：
1989-05-11
影响因子：
14.9
通讯作者：
CHINNADURAI, G
中科院分区：
文献类型：
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作者：
KUPPUSWAMY, M;SUBRAMANIAN, T;CHINNADURAI, G
HIV-1的tat基因是HIV长末端重复序列（LTR）基因表达的有效反式激活因子。为了确定HIV-1的tat基因产物（tat）的功能重要区域，构建了菌株SF2 tat编码区内的缺失、连接子插入和单氨基酸替代突变体。通过测量与HIV-LTR相关的细菌氯霉素乙酰转移酶（CAT）基因的表达来评估这些突变对反式激活的影响。这些研究表明，位于n端56个氨基酸区域内的蛋白质的四个不同结构域对Tat功能至关重要。除了必需结构域之外，在氨基酸残基58和66之间还绘制了一个增强必需区域活性的辅助结构域。其中一个基本结构域位于n端20个氨基酸区域。其他三个基本结构域在HIV-1和HIV-2以及猴免疫缺陷病毒（SIV）的不同毒株中高度保守。在保守结构域中，一个包含7个Cys残基，几个Cys残基的单氨基酸取代表明它们是Tat功能所必需的。第二个保守结构域包含一个Lys X Leu Gly Ile X Tyr基序，其中Lys残基是反式激活所必需的，其他残基是部分必需的。第三保守域是强碱性的，似乎起着双重作用。缺乏该结构域的突变体缺乏反式激活和Tat对细胞核和核仁的有效靶向。四个基本结构域和辅助结构域的结合使101个氨基酸的Tat蛋白具有接近完全的活性。
The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reported gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutant lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.