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The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is a viral accessory protein related to, but distinct from, the Vpr protein of HIV-1. Vpx is packaged into virions and, as a component of the viral preintegration complex (PIC), Vpx is required for efficient virus replication in nondividing cells. Therefore, the localization of Vpx in cells is dynamic and dependent upon discrete domains of the protein. Expressed in the absence of other viral proteins, Vpx localizes to the nucleus of cells. However, if expressed with the Gag protein of HIV-2, Vpx localizes to the plasma membrane of cells. To further understand the regulation of Vpx localization, we fused regions of Vpx to β-galactosidase to identify regions of the protein sufficient to mediate nuclear localization. The minimal transferable region of Vpx that conferred nuclear localization in these assays was aa 65 to 72. Alanine substitution of K68 and R70 in a GFP-Vpx construct abolished nuclear localization, suggesting that the basic residues in this region are important for nuclear import. Analysis of the membrane transport of several GFP-Vpx alanine mutants demonstrated that while separable, the domains of Vpx required for nuclear localization are not distinct from the domains required for membrane transport. The results of heterokaryon shuttling assays indicated that Vpx is not a shuttling protein; however, HIV-2 Vpr did shuttle similar to HIV-1 Vpr.