Full Peptide Synthesis, Purification, and Characterization of Six Tat Variants

Abstract

AIDS in Africa is characterized by the equal distribution of mortality between the two genders because of highly virulent human immunodeficiency virus type 1 (HIV-1) strains. The viral protein Tat trans-activates viral gene expression and is essential for HIV-1 replication. We chemically synthesized six different Tat proteins, with sizes ranging from 86 to 101 residues, from HIV-1 isolates located in different parts of the world including highly virulent African strains. Protein purification, mass spectroscopy, and amino acid analysis showed that the synthesis was successful in each case but with different yields. We show that all have the ability to bind the HIV long terminal repeat (LTR) RNA trans-activation response element (TAR) region, involved in Tat-mediated trans-activation, but structural heterogeneities are revealed by circular dichroism. These Tat synthetic proteins cross membranes but differ in their ability to trans-activate an HIV LTR-reporter gene in stably transfected HeLa cells. Two Tat proteins from virulent African HIV-1 strains were much more active than those from Europe and the United States. The interferon-induced kinase (PKR), involved in cell antiviral defense, phosphorylates only Tat variants corresponding to less or nonvirulent HIV-1 isolates. Our results indicate that the high virulence of some African HIV-1 strains could be related to Tat activity.The HIV 1 encodes regulatory proteins such as Tat, which profoundly affect the course of viral gene expression in infected cells. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (1, 2). This function is generally associated with the ability of Tat to bind the nascent leader RNA hairpin TAR located at the 5Ј-end of all HIV-1 mRNAs (3). Tat presents a highly conserved basic region that can adopt an extended structure to fit into the TAR major groove (4). Furthermore, this basic region provides for Tat the capacity to cross membranes (5, 6).However, the role of Tat in the HIV cycle is more than to facilitate the elongation of HIV mRNA. Tat is required for efficient HIV-1 reverse transcription (7). Activities of different cellular kinase is modulated by Tat, which results in the activation of viral transcription in infected cells (8,9). Tat protein is released from HIV-1-infected cells and can be detected in sera from HIV-1-infected patients (10). Extracellular Tat may account for the decrease of the host immune response and cellular disorders connected to AIDS pathology. Tat in synergy with a basic fibroblast growth factor (bFGF) is involved in the induction of Kaposi's sarcoma lesions (11). Tat is able to repress the major histocompatibility complex (MHC) class I genes and provides the virus with a mechanism to evade the host immune response (12). Finally, Tat participates in the induction of apoptosis in lymphocytes and contributes to the depletion of the CD4ϩ T cells in AIDS (10, 13).One of the primary cellular responses to viral infection is the production of interferon. The double-stranded RNA-dependent...

Tat HIV-1 Primary and Tertiary Structures Critical to Immune Response Against Non-homologous Variants

“…Synthesis of Tat Oyi and Tat Eli were described previously (31). The synthesis of Tat HXB2, 92Br, CM240, and Ug11RP were carried out according to the same procedure.…”

“…Transactivation Assay with HIV LTR Transfected HeLa Cells-Functional transactivation by HIV-1 Tat variants were assessed using HeLa P4 cells as previously described (31). These HeLa cells have a bacterial lacZ gene under the control of the HIV LTR, and cytoplasmic accumulation of ␤-galactosidase reveals the presence of active Tat.…”

“…The concentration of antigen was 2 g/ml diluted in phosphate buffer 100 mM pH 6. Under these conditions the Tat protein adopts tertiary active structure (31). Immunoplates were coated with 100 l/well antigen or phosphate buffer and were incubated overnight at 4°C.…”

The Glutamine-rich Region of the HIV-1 Tat Protein Is Involved in T-cell Apoptosis

“…Protein Synthesis, Purification, and Characterization-The Tat proteins were assembled according to the method of Barany and Merrifield (50) on (4-(hydroxymethyl)phenoxymethyl)copolystyrene (1% divinylbenzene)-preloaded resin (0.5-0.65 mmol; PerkinElmer Life Sciences) on an ABI 433A automated synthesizer (PerkinElmer Life Sciences) as described previously (51). Purification was carried out using a Beckman high-pressure liquid chromatography (HPLC) apparatus with a Beckman C 8 reverse-phase column (10 ϫ 150 mm).…”

“…The trans-activation function of the HIV-1 Tat variants was tested using the human HeLa P4 cells as described previously (51). Cells (2 ϫ 10 5 /well) were incubated in 24-well plates (Falcon) at 37°C and 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen) and 100 g/ml neomycin.…”