Abstract
BackgroundThe HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002). In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.ResultsOur data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.ConclusionsVpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.
Highlights
The Human immunodeficiency virus 1 (HIV-1) genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1induced apoptosis
Analysis of Vpr sequence and/or structural determinants implicated in the interaction with di-W-containing peptides We have previously used a genetic selection system in S. cerevisiae budding yeast and selected a panel of di-W-containing GST-peptides that inhibit Vpr-mediated yeast growth arrest function presumably through their ability to bind HIV-1 Vpr [38]
We further investigated the molecular mechanism of this inhibition using a newly selected GST-fused di-W peptide WWSFKSV (GST-B4), which displayed an enhanced ability to bind Vpr and inhibit its growth arrest activity in budding yeast (Fig. 1A and 1B)
Summary
The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1induced apoptosis. Virion-associated Vpr has been shown to act early in viral infection as a facilitator of HIV-1 preintegration complex (PIC) entry through the limiting nuclear pore This activity of Vpr is thought to be responsible for Vpr's ability to enhance HIV-1 replication in nondividing cells, most notably in terminally differentiated macrophages [3,4,5]. Expression of Vpr induces a G2 cell cycle arrest, which is thought to indirectly enhance viral replication by increasing transcription from the HIV-1 long terminal repeat (LTR) [6,7]. Deletion mapping studies showed that the C-terminal 33 amino acids, including the H(S/ F)RIG motif, contributed to this cytostatic effect [15,18] This region has been implicated in Vprmediated cell-cycle dysregulation in mammalian and S. Vpr cytostatic effect observed in S. cerevisiae has been proposed to result from gross mitochondrial dysfunction [17] and/or cytoskeletal defects [16], rather than a cell cycle G2 arrest
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