Structural and functional characterization of human immunodeficiency virus tat protein.

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and 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。     

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 Raf-1 cysteine rich domain: Requirement for a cluster of basic aminoacids for interaction with phosphatidylserine

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R¹⁴³, K¹⁴⁴, and K¹⁴⁸ appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Role of basic residues in the subgroup-determining region of the subgroup A avian sarcoma and leukosis virus envelope in receptor binding and infection.

Receptor specificity in avian sarcoma and leukosis viruses (ASLV) maps to the central region of the envelope surface protein, SU. Two hypervariable regions, hr1 and hr2, within this region of SU are the principal determinants of receptor specificity. The cellular receptor for subgroup A ASLV, Tva, utilizes a 40-residue, acidic, cysteine-rich sequence for viral binding and entry. This domain in Tva is closely related to the ligand-binding domain of the low-density lipoprotein receptor (LDLR). Ligands bind to LDLR via the interaction of clustered basic residues in the ligand with the acidic cysteine-rich domains of the receptor. Analysis of the ASLV envelope sequences revealed a cluster of basic residues within hr2 that is unique to the subgroup A viruses, suggesting a possible role for these residues in receptor recognition. Therefore, the effects of altering these basic residues on subgroup A envelope expression, receptor binding, and infectivity were examined. Most of the mutant proteins were transported to the cell surface and processed normally. Receptor binding was diminished approximately 50% by alanine substitution at amino acid R213 or K227, whereas substitution by alanine at R210, R223, or R224 had no effect. However, when coupled with mutations at R213 or K227, changes at R223,R224 reduced envelope binding by 90%. Mutation of all five basic residues abrogated receptor binding. The effect of the hr2 mutations on ASLV envelope-mediated infection did not parallel the effect on receptor binding. Residues 210, 213, 223, and 224 were important for efficient infection, while mutations at residue 227 had little effect on infectivity. These results demonstrate that the basic residues in the ASLV envelope have roles in both receptor recognition and post-receptor binding events during viral entry.     

Feline immunodeficiency virus ORF-Ais required for virus particle formation and virus infectivity

The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.     

Trans-dominant Tat mutants with alterations in the basic domain inhibit HIV-1 gene expression

The Tat protein of the human immunodeficiency virus type 1 (HIV-1) is required for efficient viral gene expression. By means of mutational analyses, several domains of the Tat protein that are required for complete activation of HIV-1 gene expression have been defined. These include an amino-terminal activating domain, a cysteine-rich dimerization domain, and a basic domain important in the binding of Tat to the trans-activation response element (TAR) and in Tat nuclear localization. Recently, we described a mutation, known as delta tat, which resulted in a protein with a truncated basic domain. This protein had a "trans-dominant" phenotype in that it inhibited wild-type Tat activation of the HIV-1 LTR. To further characterize the requirements for generating a Tat trans-dominant phenotype, we constructed a variety of Tat proteins with truncations or substitutions in the basic domain. A number of these proteins showed a trans-dominant phenotype. These Tat mutants also inhibited activation of the HIV-1 LTR by a protein composed of Tat fused to the prokaryotic R17 (phage MS2) RNA-binding protein in which the R17 recognition element was inserted in the HIV-1 LTR in place of TAR. Thus, an intact TAR element was not required for this inhibition. We also studied the cellular localization of Tat and a trans-dominant Tat mutant by means of immunofluorescence staining with the use of antibodies reactive to different domains of the Tat protein. The results indicated that Tat becomes localized predominantly in the nucleus both in the presence and absence of the trans-dominant Tat construct, suggesting that the trans-dominant mutant does not inhibit Tat nuclear localization. These studies further define the requirements for the creation of trans-dominant Tat mutants, and suggest that the mechanism of trans-dominant Tat inhibition may be either the formation of an inactive complex between wild-type and mutant Tat or sequestration of cellular factors involved in regulating HIV-1 gene expression.     

Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm.

We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.     

Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells

Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.