Abstract
The virion-associated protein of human immunodeficiency virus, type 1 (HIV-1), Vpr, is a small protein with 96 amino acid residues that has the ability to modulate transcription of HIV-1 long terminal repeat (LTR) promoter activity and affects several cellular functions. In this study we have employed molecular approaches to further investigate the mechanism by which Vpr exerts its regulatory effect upon the LTR. We show that by structural and functional interaction with Tat, a potent viral regulatory protein, Vpr synergistically enhances the transcriptional activity of the HIV-1 LTR. Because Tat utilizes cyclin T1 and its partner, CDK9 to elevate the level of transcription from the LTR, we examined the cooperativity between Vpr, Tat, and cyclin T1/CDK9 on viral gene transcription. Results from co-transfection studies indicated superactivation of LTR by Tat and cyclin T1/CDK9 in the presence of wild type Vpr. This activation was not observed with the R73S mutant of Vpr, which contains arginine to serine transition at residue 73. Interestingly, expression of R73S mutant in cells exerts a negative effect on the observed superactivation of the LTR by Tat, cyclin T1/CDK9, and wild type Vpr. Results from protein-protein interaction studies indicated that Vpr is associated with both Tat and cyclin T1 in cells expressing these proteins. Use of deletion mutant proteins in binding studies revealed that the binding sites for Tat and Vpr within cyclin T1 are distinct and that association of these two viral proteins with cyclin T1 is independent from each other. These observations suggest a working model on the cooperative interaction of Vpr with viral and cellular proteins and its involvement in control of viral gene transcription and replication. Moreover identification of R73S mutant of Vpr provides a new therapeutic avenue for controlling HIV-1 gene transcription and replication in the infected cells.
Highlights
Vpr represents one of the HIV-11 accessory gene products which is composed of 96 amino acid residues
Coexpression of Vpr and Tat led to a synergistic activation of viral transcription, suggesting that these two viral proteins cooperate with each other to enhance the function of the HIV-1 long terminal repeat (LTR)
We determined whether enhanced activation of HIV-1 LTR by Tat and Vpr is mediated through direct interaction between Tat and Vpr proteins
Summary
Vpr represents one of the HIV-11 accessory gene products which is composed of 96 amino acid residues. Vpr associates with a number of cellular factors including p300 and TFIIB and modulates the activity of the HIV-1 LTR [13, 15, 16] Some of these factors cooperate with the viral potent transcriptional activator, Tat [17, 18]. Because Tat and Vpr are involved in the activation of the HIV-1 LTR through either the interaction with several common cellular regulatory proteins [13, 15,16,17,18] and/or binding to an RNA molecule [14, 27], we focused our attention on the structural and functional interplay between these two viral proteins. In this study we provide evidence for the physical interaction of Vpr with Tat and demonstrate that cooperativity between Tat, This paper is available on line at http://www.jbc.org
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