Tiley L S, Brown P H, Cullen B R
Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710.
Virology. 1990 Oct;178(2):560-7. doi: 10.1016/0042-6822(90)90354-t.
Human immunodeficiency virus type 1 (HIV-1) encodes a transcriptional trans-activator, termed Tat, that is absolutely required for viral replication in vitro. By analogy to other known transcription factors, it has been suggested that the HIV-1 Tat protein may contain discrete protein domains that determine sequence specificity and transcriptional activation potential. Here, we report the use of site-directed mutagenesis to examine the functional significance of two candidate activation domains within Tat. A 12 amino acid sequence adjacent to the N-terminus of the Tat protein, which includes a proposed acidic amphipathic alpha-helix activation motif, was found to contribute to, but be dispensable for, Tat function in vivo. In contrast, the integrity of a second potential Tat activation motif, centered on a lysine residue at position 41, was found to be essential for Tat function. However, Tat proteins mutated in this area displayed a fully recessive negative phenotype. Therefore, neither of these two regions of the Tat protein appear to be discrete activation domains. We conclude that previous attempts to categorize Tat as a modular transcription factor have not succeeded and suggest that the functional organization of this complex trans-activator remains to be defined.
1型人类免疫缺陷病毒（HIV-1）编码一种转录反式激活因子，称为Tat，它是病毒在体外复制所绝对必需的。类比于其他已知的转录因子，有人提出HIV-1 Tat蛋白可能包含决定序列特异性和转录激活潜能的离散蛋白结构域。在此，我们报告利用定点诱变来研究Tat内两个候选激活结构域的功能意义。发现Tat蛋白N端附近的一段12个氨基酸的序列，其中包括一个推测的酸性两亲性α螺旋激活基序，对Tat在体内的功能有贡献，但并非必不可少。相反，以第41位赖氨酸残基为中心的第二个潜在Tat激活基序的完整性对于Tat功能至关重要。然而，在该区域发生突变的Tat蛋白表现出完全隐性负表型。因此，Tat蛋白的这两个区域似乎都不是离散的激活结构域。我们得出结论，先前将Tat归类为模块化转录因子的尝试并未成功，并表明这种复杂反式激活因子的功能组织仍有待确定。
