Journal of Molecular Biology
Regular articleHuman immunodeficiency virus type 1 vpr protein transactivation function: mechanism and identification of domains involved1
Introduction
Extensive efforts have recently been made to decipher the function and mechanism of action of human immunodeficiency virus (HIV) accessory proteins. Of particular interest is Vpr, a 96 amino acid residue product that is well conserved among HIV and simian immunodeficiency virus (SIV) subtypes (Tristem et al., 1990). This protein is expressed from a singly spliced mRNA whose expression is dependent on Rev (Schwartz et al., 1991). The role of Vpr in the HIV-1 infection cycle is not clear, although an array of associated phenotypes has been identified over the years Subbramanian and Cohen 1994, Trono 1995, Emerman 1996. An important property of Vpr is its ability to be incorporated into viral particles (Cohen et al., 1990a), suggesting a role in the early steps of infection. Vpr has been shown to increase viral production. This effect was first observed in transformed T-cells (Cohen et al., 1990b) but is more pronounced in non-dividing cells such as macrophages Westervelt et al 1992, Connor et al 1995, Subbramanian et al 1998a. In non-dividing cells, Vpr appears to be involved in the nuclear translocation of the pre-integration complex (Heinzinger et al., 1994) but seems also to influence post-integration events Connor et al 1995, Subbramanian et al 1998b.
A recently identified biological activity associated with Vpr is its ability to prevent the passage of cells through mitosis at the G2 stage of the cell-cycle Rogel et al 1995, Jowett et al 1995. Indeed, Vpr expression results in the inactivation of the cyclin-dependent kinase p34cdc2 and of its upstream regulator cdc25 He et al 1995, Re et al 1995. The exact target of Vpr in the regulation of the cell-cycle in vivo is not known. The cell-cycle arrest phenomenon has been observed in various yeast strains and prokaryotic cells, indicating that the pathway targeted by Vpr is conserved among various cell types Bodeus et al 1997, Macreadie et al 1995, Zhao et al 1996, Zhang et al 1997. In addition, the cell-cycle arrest function is well conserved among HIV and SIV Vpr alleles and is relatively species-specific Fletcher et al 1996, Planelles et al 1996, Stivahtis et al 1997. Eventually, Vpr-induced G2 arrest leads to cell death by apoptosis Stewart et al 1997, Subbramanian et al 1998a, Yao et al 1998. Progress in elucidating the functional role of Vpr-mediated cell-cycle arrest during HIV-1 infection has been made recently. Accumulating evidence indicates that G2 arrest creates a favorable environment for maximizing viral expression and production Goh et al 1998, Yao et al 1998.
We have previously shown that Vpr is a moderate transactivator of the HIV-1 long terminal repeat (LTR) as well as of other heterologous promoters such as the murine leukemia virus (MLV) SL3-3 LTR (Cohen et al., 1990b). These studies as well as those by Agostini et al. (1996) have demonstrated that the transactivation ability of Vpr does not depend on specific responsive promoter sequences. Moreover, Vpr was shown to interact with several cellular proteins Zhao et al 1994, Refaeli et al 1995, Wang et al 1995, Agostini et al 1996, Bouhamdan et al 1996, Withers-Ward et al 1997. Studies by Wang et al. (1995)have shown in vitro an association of Vpr with the promoter-bound transcription factor Sp1. This interaction was further supported in in vitro transcription assays, where Vpr was able to transactivate a minimal LTR containing only the TATA box and Sp1 binding sites. Agostini et al. (1996) have also identified TFIIB, a protein that is part of the basal machinery of transcription, as a cellular partner for Vpr in vitro. An interaction of Vpr with a 41 kDa (RIP-1) protein that associates with the glucocorticoid receptor has been reported (Refaeli et al., 1995). Taking into account the ability of Vpr to interact with these cellular proteins, transactivation could be the result of interactions with specific transcription factors and/or the basal transcription machinery. In this study, we have investigated the mechanism of Vpr-mediated transactivation. We report here that Vpr stimulates LTR-directed reporter gene expression in vivo by increasing mRNA levels. Vpr was found to transactivate promoters only when their basal expression reached a minimal level. Using mutagenic analysis, we provide evidence confirming that Vpr-mediated transactivation of gene expression and cell-cycle arrest are functionally related. Moreover, the capacity of Vpr to transactivate does not require the same determinants as those involved in viral incorporation or protein nuclear localization.
Section snippets
Vpr transactivates gene expression by increasing mRNA levels
Although Vpr increases gene expression, the mechanism by which Vpr mediates this effect in vivo has not been clearly determined. We used the ability of Vpr to increase reporter gene expression driven by either the HIV-1 LTR or the heterologous MLV SL3-3 LTR to clarify its mechanism of action. Briefly, Jurkat cells were co-transfected with either a plasmid expressing Vpr from the Bru strain of HIV-1 (SVCMVBruR) or an ATG-minus version of this construct (SVCMVBruRATG−), and a plasmid encoding the
Discussion
In this study we have investigated the mechanism by which HIV-1 Vpr transactivates homologous as well as heterologous promoter-directed gene expression. Our results provide evidence indicating that Vpr expression increases the levels of mRNA transcribed from the HIV-1 and MLV SL3-3 LTRs in a transfected Jurkat T cell line. From our data, we cannot distinguish whether the modulation of mRNA levels mediated by Vpr is the result of an effect at the level of transcription initiation or mRNA
Cell culture and DNA transfection
The Jurkat CD4+ T-cell line was obtained from the ATCC and maintained in RPMI (GIBCO BRL, Burlington, Canada) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillin-streptomycin (P/S). HeLa cells were maintained in DMEM (GIBCO BRL, Burlington, Canada) supplemented with 10% FBS and 1% P/S. Jurkat cells were transfected by the DEAE-dextran method as described (Cohen et al., 1990b). HeLa cells were transfected using the calcium phosphate method (Cohen et al., 1990b).
Plasmids
The
Acknowledgements
We thank Dr Daniel Celander for providing the mutated pSU3CAT constructs, Christian Lemaire for his helpful discussions on semi-quantitative PCR, and Ramu Subbramanian for the design of the Vpr mutants as well as helpful discussions, and Robert Lodge for critical reading of the manuscript. J.F. is a recipient of a studentship from the National Health Research and Development Program (NHRDP) of Health and Welfare Canada. É.A.C. is a recipient of a Medical Research Council of Canada (MRC)
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Edited by J. Karn