Echetebu C O, Rhim H, Herrmann C H, Rice A P
Division of Molecular Virology, Baylor College of Medicine, Houston, Texas 77030.
J Acquir Immune Defic Syndr (1988). 1994 Jul;7(7):655-64.
The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) Tat proteins Tat-1 and Tat-2 stimulate transcription of the viral long terminal repeat (LTR) sequences and are required for efficient viral replication. A class of mutant Tat proteins, termed "transdominant mutants," has been described that possesses relatively low transactivation activity, yet is able to inhibit the function of wild-type Tat. These mutant proteins contain a nonfunctional TAR RNA-binding domain but apparently retain a functional activation domain. A potential limitation for therapeutic use of transdominant mutants described to date is their low but significant basal level of transactivation for the HIV-1 or HIV-2 LTRs. In order to make an improved transdominant mutant, we have constructed Tat-2 proteins that contain mutations in four contiguous arginines at residues 81 to 84 in the RNA-binding domain. Using purified proteins and in vitro RNA-binding assays, we verified that these mutant Tat-2 proteins are defective for TAR RNA binding. We also verified that these mutant Tat-2 proteins bind to a cellular protein kinase in vitro that we have previously shown to bind specifically to the Tat-1 and Tat-2 activation domain. Using plasmid cotransfection assays, we compared the phenotypes of these mutant Tat-2 proteins with the most potent Tat-1 transdominant mutant described to date. One Tat-2 mutant, named "R81-84A," was found to be equivalent to the Tat-1 mutant in ability to inhibit wild-type Tat transactivation of HIV-1 and HIV-2 LTRs. Moreover, the R81-84A mutant possessed a significantly lower basal level of transactivation than the Tat-1 mutant. The R81-84A Tat-2 mutant is therefore a promising reagent for future development as an anti-HIV agent. Additionally, our results suggest that wild-type Tat-2 transactivation of the HIV-2 LTR is especially sensitive to inhibition by transdominant mutants.
1型和2型人类免疫缺陷病毒（HIV-1和HIV-2）的反式激活蛋白Tat-1和Tat-2可刺激病毒长末端重复序列（LTR）的转录，是病毒高效复制所必需的。已描述了一类突变型Tat蛋白，称为“反式显性突变体”，其具有相对较低的反式激活活性，但能够抑制野生型Tat的功能。这些突变蛋白含有无功能的TAR RNA结合结构域，但显然保留了功能性激活结构域。迄今为止所描述的反式显性突变体用于治疗的一个潜在限制是它们对HIV-1或HIV-2 LTRs具有较低但显著的基础反式激活水平。为了制备改良的反式显性突变体，我们构建了在RNA结合结构域中第81至84位残基的四个连续精氨酸处含有突变的Tat-2蛋白。使用纯化的蛋白和体外RNA结合试验，我们证实这些突变型Tat-2蛋白在TAR RNA结合方面存在缺陷。我们还证实这些突变型Tat-2蛋白在体外与一种细胞蛋白激酶结合，我们之前已证明该激酶可特异性结合Tat-1和Tat-2激活结构域。使用质粒共转染试验，我们将这些突变型Tat-2蛋白的表型与迄今为止所描述的最有效的Tat-1反式显性突变体进行了比较。发现一种名为“R81-84A”的Tat-2突变体在抑制HIV-1和HIV-2 LTRs的野生型Tat反式激活能力方面与Tat-1突变体相当。此外，R81-84A突变体的基础反式激活水平明显低于Tat-1突变体。因此，R81-84A Tat-2突变体是未来作为抗HIV药物开发的有前景的试剂。此外，我们的结果表明，HIV-2 LTR的野生型Tat-2反式激活对反式显性突变体的抑制特别敏感。
