Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

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.     

Mutational analysis of HIV-1 Tat minimal domain peptides: identification of trans-dominant mutants that suppress HIV-LTR-driven gene expression

The HIV-1 Tat protein is a potent trans-activator essential for virus replication. We reported previously that HIV-1 Tat peptides containing residues 37-48 (mainly region II), a possible activating region, and residues 49-57 (region III), a nuclear targeting and putative nucleic acid binding region, possess minimal but distinct trans-activator activity. The presence of residues 58-72 (region IV) greatly enhances trans-activation. We postulate that Tat mutant peptides with an inactive region II and a functional region III can behave as dominant negative mutants. We synthesized minimal domain peptides containing single amino substitutions for amino acid residues within region II that are conserved among different HIV isolates. We identify four amino acid residues whose substitution within Tat minimal domain peptides leads to defects in transactivation. Some of these mutants are trans-dominant in several peptide backbones, since they strongly inhibit trans-activation by wild-type Tat protein added to cells or expressed from microinjected plasmid. Significantly, trans-activation of integrated HIV-LTRCAT is blocked by some trans-dominant mutant peptides. These results suggest an attractive approach for the development of an AIDS therapy.     

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.     

Structural analysis of wild-type and mutant human immunodeficiency virus type 1 Tat proteins.

We expressed the human immunodeficiency virus type 1 transactivator protein, Tat, in the wheat germ cell-free translation system and found it to exist as a monomer. The first coding exon (residues 1 to 72) of wheat germ-expressed Tat was resistant to trypsin digestion, indicating that it is a highly folded, independently structured protein domain. Several mutant Tat proteins were dramatically more sensitive to trypsin than the wild type was, suggesting that their reduced transactivation activities are the result of destabilized structures. Mutant proteins with single-amino-acid substitutions were also identified that had reduced transactivation activities but wild-type structures in the trypsin assay. These mutants clustered in two regions of Tat, at acidic residues 2 and 5 in the amino terminus and between residues 18 and 32. These mutants, wild type in structure but reduced in activity, identify residues in the wild-type protein that may directly contact other molecules during Tat function.     

Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor.

Efficient replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) requires the virus transactivator proteins known as Tat. In order to understand the molecular mechanisms involved in Tat transactivation, it is essential to identify the cellular target(s) of the Tat activation domain. Using an in vitro kinase assay, we previously identified a cellular protein kinase activity, Tat-associated kinase (TAK), that specifically binds to the activation domains of Tat proteins. Here it is demonstrated that TAK fulfills the genetic criteria established for a Tat cofactor. TAK binds in vitro to the activation domains of the Tat proteins of HIV-1 and HIV-2 and the distantly related lentivirus equine infectious anemia virus but not to mutant Tat proteins that contain nonfunctional activation domains. In addition, it is shown that TAK is sensitive to dichloro-1-beta-D-ribofuranosylbenzimidazole, a nucleoside analog that inhibits a limited number of kinases and is known to inhibit Tat transactivation in vivo and in vitro. We have further identified an in vitro substrate of TAK, the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation of the carboxyl-terminal domain has been proposed to trigger the transition from initiation to active elongation and also to influence later stages during elongation. Taken together, these results imply that TAK is a very promising candidate for a cellular factor that mediates Tat transactivation.     

河南HIV感染者的HIV-1B型株tat基因的克隆及序列分析

克隆河南人免疫缺陷病毒(HIV)感染HIV-1B型株tat基因完整编码框序列,并分析比较其编码产物的序列结构特点。使用重叠PCR技术,从河南省1名HIV-1感染外周血标本中扩增出to／基因第一和第二外显子并重组为完整的tat基因序列。获得的HIV-1B病毒株tat基因,第一外显子为263bp,第二外显子为214bp。将该基因编码产物与其他HW-1株Tat蛋白经DNA软件编辑并翻译成蛋白质,使用Clustal X1．81进行多序列对比分析发现,第一外显子编码产物的3个保守区域的氨基酸组成大致相同,只有少数氨基酸存在差异。由于Tat蛋白不同病毒株间有高度保守的Cys富集区、核心区和碱性氨基酸富集区,tat基因的克隆为研究其功能并以其为靶点设计和筛选抗艾滋病的药物奠定了基础。     

Cutting edge: a short polypeptide domain of HIV-1-Tat protein mediates pathogenesis.

HIV-1 encodes the transactivating protein Tat, which is essential for virus replication and progression of HIV disease. However, Tat has multiple domains, and consequently the molecular mechanisms by which it acts remain unclear. In this report, we provide evidence that cellular activation by Tat involves a short core domain, Tat21-40, containing only 20 aa including seven cysteine residues highly conserved in most HIV-1 subtypes. Effective induction by Tat21-40 of both NF-kappaB-mediated HIV replication and TAR-dependent transactivation of HIV-long terminal repeat indicates that this short sequence is sufficient to promote HIV infection. Moreover, Tat21-40 possesses potent angiogenic activity, further underscoring its role in HIV pathogenesis. These data provide the first demonstration that a 20-residue core domain sequence of Tat is sufficient to transactivate, induce HIV replication, and trigger angiogenesis. This short peptide sequence provides a potential novel therapeutic target for disrupting the functions of Tat and inhibiting progression of HIV disease.