Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.

Human cyclin T1 (hCycT1), a major subunit of the essential elongation factor P-TEFb, has been proposed to act as a cofactor for human immunodeficiency virus type 1 (HIV-1) Tat. Here, we show that murine cyclin T1 (mCycT1) binds the activation domain of HIV-1 Tat but, unlike hCycT1, cannot mediate Tat function because it cannot be recruited efficiently to TAR. In fact, overexpression of mCycT1, but not hCycT1, specifically inhibits Tat-TAR function in human cells. This discordant phenotype results from a single amino acid difference between hCycT1 and mCycT1, a tyrosine in place of a cysteine at residue 261. These data indicate that the ability of Tat to recruit CycT1/P-TEFb to TAR determines the species restriction of HIV-1 Tat function in murine cells and therefore demonstrate that this recruitment is a critical function of the Tat protein.     

树鼩细胞周期蛋白T1 cDNA的克隆和序列分析

树鼩细胞不能感染HIV-1,但支持HIV-1进入靶细胞后的转录,可能是因为树鼩细胞周期蛋白T1(tsCycT1)能结合HIV-1的Tat蛋白。通过设计引物,用RT—PCR技术,获得全长为2175bp tsCycT1基因的cDNA。其核苷酸序列与人的CycT1(hCycT1)基因的cDNA有92．6％的相似性;由此推导出的氨基酸序列有94．1％相似性。其中,hCycT1和tsCycT1氨基端的1—272个氨基酸的相似性高达98．8％,氨基酸第261位点为半胱氨酸。这些结果提示,tsCycT1会和HIV-1的Tat结合,形成高亲和的、锌依赖的复合物,支持HIV-1转录。     

Cyclin T: three forms for different roles in physiological and pathological functions

Cyclins are members of family of proteins involved in the cell cycle regulation. They are regulatory subunits of complexes with proteins called cyclin-dependent kinases (CDKs). There are three forms of cyclin T: cyclin T1, cyclin T2a, and T2b. All cyclin T contain an N-terminal "cyclin homology box," the most conserved region among different members of the cyclin family that serves to bind CDK9. In addition to the N-terminal cyclin domain, cyclin T contains a putative coiled-coil motif, a His-rich motif, and a C-terminal PEST sequence. The CDK9/cyclin T complex is able to activate gene expression in a catalytic-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. In addition, only cyclin T1 supports interactions between Tat and TAR. The interaction of Tat with cyclin T1 alters the conformation of Tat to enhance the affinity and specificity of the Tat:TAR interaction. On the other hand, CDK9/cyclin T2 complexes are involved in the regulation of terminal differentiation in muscle cells.     

Mutational analysis of the conserved cysteine-rich region of the human immunodeficiency virus type 1 Tat protein.

The Tat transactivator protein of human immunodeficiency virus type 1 contains a highly conserved cysteine-rich region, containing seven cysteines from residues 22 through 37. To investigate the importance of noncysteine residues in this region of the Tat protein, we have carried out a mutational analysis, in most cases substituting a single alanine for the wild-type noncysteine residue. Alanine substitution of residue 23, 24, 46, or 47 had no effect on Tat activity in plasmid transfection assays. In contrast, alanine substitutions of all eight noncysteines analyzed, from residues 26 through 41, significantly reduced the activity of the Tat protein, in some cases as drastically as mutations in cysteine residues. The results demonstrate that the precise sequence of the cysteine-rich region is crucial for a fully functional Tat protein.     

Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells.

Levels of trans activation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) by the virally encoded transactivator Tat show marked species-specific differences. For example, levels of transactivation observed in Chinese hamster ovary (CHO) rodent cells are 10-fold lower than those in human cells or in CHO cells that contain the human chromosome 12. Thus, the human chromosome 12 codes for a protein or proteins that are required for optimal Tat activity. Here, the function of these cellular proteins was analyzed by using a number of modified HIV-1 LTRs and Tats. Neither DNA-binding proteins that bind to the HIV-1 LTR nor proteins that interact with the activation domain of Tat could be implicated in this defect. However, since species-specific differences were no longer observed with hybrid proteins that contain the activation domain of Tat fused to heterologous RNA-binding proteins, optimal interactions between Tat and the trans-acting responsive RNA (TAR) must depend on this factor(s).