Kuppuswamy M, Subramanian T, Srinivasan A, Chinnadurai G
Institute for Molecular Virology, St Louis University School of Medicine, MO 63110.
Nucleic Acids Res. 1989 May 11;17(9):3551-61. doi: 10.1093/nar/17.9.3551.
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) reporter 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. Mutants 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.
HIV-1的tat基因是HIV长末端重复序列（LTR）基因表达的强效反式激活因子。为了确定HIV-1 tat基因（Tat）产物的功能重要区域，构建了SF2株Tat编码区域内的缺失、接头插入和单氨基酸取代突变体。通过测量与HIV-LTR相连的细菌氯霉素乙酰转移酶（CAT）报告基因的表达，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个氨基酸区域内的该蛋白的四个不同结构域对Tat功能至关重要。除了这些必需结构域，一个增强必需区域活性的辅助结构域也已定位在氨基酸残基58和66之间。其中一个必需结构域位于N端20个氨基酸区域。其他三个必需结构域在HIV-1和HIV-2的各种毒株以及猴免疫缺陷病毒（SIV）中高度保守。在这些保守结构域中，一个含有七个半胱氨酸残基，几个半胱氨酸残基的单氨基酸取代表明它们对Tat功能至关重要。第二个保守结构域包含一个Lys-X-Leu-Gly-Ile-X-Tyr基序，其中Lys残基对反式激活至关重要，其他残基部分必需。第三个保守结构域具有强碱性，似乎发挥双重作用。缺乏该结构域的突变体在反式激活以及将Tat有效靶向细胞核和核仁方面存在缺陷。这四个必需结构域和辅助结构域的组合促成了101个氨基酸的Tat蛋白所观察到的近乎完全的活性。
