Identification of the nuclear localization signal of human immunodeficiency virus type 2 Vpx.
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 beta-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 K(68) and R(70) 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.
Belshan
Michael
M
Departments of Medicine, Pathology, and Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ratner
Lee
L
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015498:HIV-2 / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007739:L Cells; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals / Q000032:analysis*; D012191:Retroviridae Proteins / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase
D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D012191:Retroviridae Proteins; C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D000409:Alanine