11th International AIDS Conference Vancouver, British Columbia — July 7-12, 1996

Int Conf AIDS 1996 Jul 7-12; 11:22 (abstract no. LB.A.6004)
Takaori-Kondo A, de Noronha C, McEntee M, Greene W; Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA. Fax: (415) 826-15149826-1817. E-mail: akifumi_takaori-kondo.giv@quickmail.ucsf.edu.

OBJECTIVE: To delineate the molecular mechanism by which HIV-1 Vpr induces G2 cell cycle arrest.

METHODS AND RESULTS: To identify cellular proteins that might functionally associate with Vpr, a yeast-two hybrid screen was performed using Vpr as "bait". One specific Vpr-interacting cellular protein corresponded to the regulatory or A subunit of protein phosphatase 2A (PP2A-A). This interaction was particularly intriguing since the PP2A holoenzyme has been shown to oppositely regulate the cdc25 tyrosine phosphatase and Wee-1 tyrosine kinase activities that determine the phosphorylation status and activation status of cdc2, a kinase that is essential for progression through the G2 cell cycle checkpoint. To independently test the physical interaction of PP2A and Vpr, glutathione S transferase (GST) pulldown experiments were performed. GST-Vpr fusion proteins specifically bound and retained hemagglutinin (HA) epitope tagged PP2A-A present in transfected cell lysates, but failed to interact with comparably prepared HA-phosphatidylinositol kinase. Similarly, GST-PP2A-A fusion proteins specifically bound and retained wild type Vpr. In contrast, GST-PP2A-A did not bind to functionally inactive mutants of Vpr (Vpr-R80A, Vpr-84stop) which biologically fail to produce G2 cell cycle arrest. Addition of okadaic acid, an inhibitor of protein phosphatase 2A, blocked both Vpr mediated G2 cell cycle arrest and Vpr induced hyperphosphorylation of the cdc2 kinase. Finally, in vivo reconstitution studies were performed using Vpr resistant baby hamster kidney BHK cell for transfection. While these cells fail to undergo G2 arrest in the presence of Vpr, co-transfection of the regulatory (A) and catalytic subunits of the human PP2A holoenzyme reconstituted Vpr mediated G2 cell cycle arrest underscoring the importance of this interaction.

CONCLUSIONS: The findings delineate a molecular mechanism by which HIV-1 Vpr produces G2 cell cycle arrest involving its physical interaction with the A regulatory subunit of protein phosphatase 2A. This interaction apparently augments PP2A action, leading to diminished cdc25 tyrosine phosphatase and perhaps increased Wee-1 tyrosine kinase activity. These changes in turn produce hyperphosphorylation and inactivation of the cdc2 kinase and arrest of cells in G2. These findings highlight the ability of HIV to subvert a normal cellular regulatory pathway leading to striking changes in cell cycle progression. While presently unknown, this action of Vpr may provide a more favorable intracellular environment for HIV replication or alternatively allow the virus to elude CTL mediated immune attack.

960707
LBA6004

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