Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Yao
Xiao-Jian
XJ
Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3C 3J7, Canada. yao2@cc.umanitoba.ca
Rougeau
Nicole
N
Duisit
Ghislaine
G
Lemay
Julie
J
Cohen
Eric A
EA
D001483:Base Sequence; D002455:Cell Division; D017931:DNA Primers; D016336:Gene Products, vpr / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000254:growth & development / Q000821:virology; D054325:vpr Gene Products, Human Immunodeficiency Virus
D017931:DNA Primers; D016336:Gene Products, vpr; D010446:Peptide Fragments; D054325:vpr Gene Products, Human Immunodeficiency Virus