7th Conference - Abstract 219

HIV-1 Vpr Interferes with Cell Cycle Signaling Cascades by Interacting with the Ba Subunit of Protein Phosphatase 2A (PP2A)

M. HRIMICH*, X.-J. YAO, and E. A. COHEN. Univ. de Montreal, Quebec, Canada

The Vpr protein of primate lentiviruses induces a cell cycle G2 arrest that was shown to activate transcription from the LTR resulting in increased virus production. Several studies have previously shown that Vpr mediates cell cycle arrest through an inactivation of cyclinB-p34cdc2 kinase and its upstream regulator CDC25 phosphatase. However, cdc2 and CDC25 do not appear to be direct substrates of Vpr since no direct interaction was detected. The fact that treatment with okadaic acid, an inhibitor of PP 1 and 2A, was found to overcome Vpr-mediated G2 arrest suggests the involvement of upstream regulators of cdc2. In this study, we have investigated the interaction of Vpr with the serine/threonine PP2A, a holoenzyme that plays a critical role in signaling pathways controlling cell cycle progression. PP2A consists of a heterodimer between a catalytic subunit (C) and a regulatory subunit (A) which can further associate with one of variable regulatory B subunits that confer substrate specificity. Here, we demonstrate by coprecipitation in a cell-based assay that Vpr interacts with the PP2A holoenzyme via its Ba regulatory subunit. Expression of a transdominant mutant of the PP2A regulatory subunit that binds C but not B subunits, not only interrupts Vpr-PP2A interaction, but also abrogates Vpr cell cycle arrest. Furthermore, the interaction of Vpr with the Ba subunit of PP2A is shown to be necessary for G2 arrest since C-terminal Vpr mutants that are unable to mediate G2 arrest do not interact with PP2A. Finally, using an in vitro PP2A enzymatic assay, we show that wt Vpr, but not that of cell cycle arrest-defective Vpr mutants, enhances the activity and the affinity of PP2A for an universal substrate. In conclusion, our results strongly suggest that HIV-1 Vpr mediates cell cycle arrest in G2 by targeting PP2A and positively modulating its catalytic activity toward specific substrates.

Key Words: Cell cycle arrest, PP2A, Vpr