scholarly journals Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells

2002 ◽  
Vol 83 (5) ◽  
pp. 1173-1181 ◽  
Author(s):  
Jinseu Park ◽  
Jiyoon Ryu ◽  
Kyeong-Ae Kim ◽  
Hak Joo Lee ◽  
Jae Hoon Bahn ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Western Blot ◽  
Blot Analysis ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Tat Protein ◽  
Immunodeficiency Virus

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains a high proportion of arginine and lysine residues, is responsible for highly efficient protein transduction through the plasma membrane. To identify the role of the PTD sequence motif in transduction, various deletions and substitutions were introduced into the PTD. Tat–green fluorescent protein (GFP) fusion proteins, containing various lengths of the Tat PTD, were expressed and the extent of their transduction into mammalian cells was analysed by Western blot analysis and fluorescence microscopy. Deletion analysis of PTD mapped to a nine amino acid motif (residues 49–57: RKKRRQRRR) sufficient for transduction. Further deletion of this Tat basic domain either at the N terminus or at the C terminus significantly decreased transduction efficiency. The transduction efficiencies of GFPs fused to nine consecutive lysine (9Lys–GFP) or arginine (9Arg–GFP) residues were similar to that of Tat(49–57)–GFP. The transduced proteins localized to both the nucleus and the cytosol, as assessed by confocal microscopy and Western blot analysis of subcellular fractions from transduced cells. Thus, the availability of recombinant GFP fusion proteins facilitates the simple and specific identification of protein transduction mediated by these peptide sequences. The modified PTD sequences designed in this study may provide useful tools necessary for delivering therapeutic proteins/peptides into cells.

scholarly journals Addition of a C-Terminal Cysteine Improves the Anti-Herpes Simplex Virus Activity of a Peptide Containing the Human Immunodeficiency Virus Type 1 TAT Protein Transduction Domain

2007 ◽  
Vol 51 (5) ◽  
pp. 1596-1607 ◽  
Author(s):  
Hermann Bultmann ◽  
Jeremy Teuton ◽  
Curtis R. Brandt
Keyword(s):  
Human Immunodeficiency Virus ◽  
Herpes Simplex ◽  
Antiviral Activity ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Tat Protein ◽  
C Peptide ◽  
Immunodeficiency Virus ◽  
Simplex Virus

ABSTRACT Previous studies have shown that peptides containing the protein transduction domain (PTD) of the human immunodeficiency virus tat protein (GRKKRRQRRR) were effective inhibitors of herpes simplex virus type 1 (HSV-1) entry (H. Bultmann and C. R. Brandt, J. Biol. Chem. 277:36018-36023, 2002). We now show that the addition of a single cysteine residue to the C terminus of the TAT PTD (TAT-C peptide) improves the antiviral activity against HSV-1 and HSV-2. The principle effect of adding the cysteine was to enable the peptide to inactivate virions and to induce a state of resistance to infection in cells pretreated with peptide. The TAT-C peptide acted extracellularly, immediately blocked entry of adsorbed virus, prevented VP16 translocation to the nucleus, and blocked syncytium formation and cell-cell spread. Thus, TAT-C peptides are fusion inhibitors. The induction of the resistance of cells to infection was rapid, recovered with a half-life of 5 to 6 h, and could be reinduced by peptide treatment. TAT-C bound to heparan sulfate but was a poor competitor for viral attachment. The antiviral activity depended on the net positive charge of the peptide but not on chirality, and a free sulfhydryl group was not essential for antiviral activity because TAT-C dimers were at least as effective as monomers. The unique combination of antiviral activities and low toxicity combine to make TAT-C a strong candidate for further development as a drug to block HSV infection.

scholarly journals The Human Immunodeficiency Virus Type 1 Tat Protein Up-Regulates the Promoter Activity of the Beta-Chemokine Monocyte Chemoattractant Protein 1 in the Human Astrocytoma Cell Line U-87 MG: Role of SP-1, AP-1, and NF-κB Consensus Sites

2000 ◽  
Vol 74 (4) ◽  
pp. 1632-1640 ◽  
Author(s):  
Siew Pheng Lim ◽  
Alfredo Garzino-Demo
Keyword(s):  
Human Immunodeficiency Virus ◽  
Promoter Activity ◽  
Tat Protein ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Monocyte Chemoattractant Protein ◽  
Astrocytoma Cell ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides −123 and −115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-κB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-κB site (located at −128 to −122 and −150 to −137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (−156 to −150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-κB, leading to synergistic activation of the MCP-1 promoter.

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