Yao Xiao-Jian, Rougeau Nicole, Duisit Ghislaine, Lemay Julie, Cohen Eric A
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Retrovirology. 2004 Aug 16;1:21. doi: 10.1186/1742-4690-1-21.
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-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. Clement, 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.
Our data clearly shows that integrity of N-terminal alpha-helix I (17-33) and alpha-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.
Vpr-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 alpha-helix I (17-33) and alpha-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.
HIV-1基因组编码一种高度保守的辅助基因产物Vpr，它在逆转录病毒生命周期中发挥多种功能，包括增强病毒在非分裂巨噬细胞中的复制、诱导G2期细胞周期停滞以及调节HIV-1诱导的细胞凋亡。我们之前报道了一组含双色氨酸（W）的肽的基因筛选，这些肽能够与HIV-1 Vpr相互作用并在酿酒酵母中抑制其细胞生长抑制活性（Yao, X.-J., J. Lemay, N. Rougeau, M. Clement, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816 - 48826, 2002）。在本研究中，我们对Vpr进行了诱变分析，以确定与含双W肽相互作用相关的序列和/或结构决定因素，并评估突变对酿酒酵母中Vpr诱导的细胞生长抑制活性的影响。
我们的数据清楚地表明，N端α螺旋I（17 - 33）和α螺旋III（53 - 83）的完整性对于Vpr与含双W肽的相互作用以及对出芽酵母中蛋白质诱导的细胞生长抑制作用至关重要。有趣的是，几个Vpr突变体，主要在N端和C端结构域，之前报道它们在人类细胞中细胞周期停滞或凋亡方面存在缺陷，但在酿酒酵母中仍表现出细胞生长抑制活性，并且对含双W肽的抑制作用仍然敏感。
通过双W肽与Vpr功能域（包括α螺旋I（17 - 33）和α螺旋III（53 - 83））的特异性相互作用，GST融合的双W肽可以有效抑制出芽酵母中Vpr诱导的生长停滞。此外，出芽酵母中Vpr诱导的细胞生长抑制作用的潜在机制可能与人类细胞中细胞周期改变和凋亡所涉及的机制不同。
