MUTATIONAL ANALYSIS OF CELL-CYCLE ARREST, NUCLEAR-LOCALIZATION, AND VIRION PACKAGING OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VPR

Human immunodeficiency virus type 1 Vpr is a virion-associated, regula tory protein that is required for efficient viral replication in monoc ytes/macrophages. The protein is believed to act in conjunction with t he Gag matrix protein to allow import of the viral preintegration comp lex in nondividing cells. In cells, Vpr localizes to the nucleus. Rece ntly, we showed that Vpr prevents the activation of p34(cdc2)-cyclin B . This results in arrest of Vpr-expressing cells in the G(2)/M phase o f the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to de fine the functional domains of the protein. The results showed cell cy cle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the aminoterminal alpha-helical region o f Vpr was required for nuclear localization and packaging into virions . The carboxy terminus appeared to be unnecessary for nuclear localiza tion, In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to th e nucleus. In addition, we show that simian immunodeficiency virus Vpr , but not Vpx, induces G(2) arrest. We speculate that Vpr has two site s for interaction with cellular factors: one in the alpha-helical regi on that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.