Abstract
Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (VprN17C74) contained a bona fide nuclear localization signal and it is targeted VprN17C74 to the nuclear envelope and then imported into the nucleus by importin α (Impα) alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to VprN17C74. This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM) region (which is also essential for the binding of CAS, the export receptor for Impα) in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1–mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.
Highlights
Molecular trafficking between the nucleus and the cytoplasm is tightly regulated in eukaryotic cells
A chimeric protein comprising full-length Vpr fused at the Nterminus to GST and green fluorescent protein (GFP) (,63 kDa) was constructed, which surpassed the limit for passive diffusion into the nucleus (Fig. 1A)
This study investigated the nuclear import of full-length Vpr, the human immunodeficiency virus type 1 (HIV-1) accessory protein, using an in vitro nuclear import assay with digitonin-permeabilized HeLa cells and a pull-down assay
Summary
Molecular trafficking between the nucleus and the cytoplasm is tightly regulated in eukaryotic cells. The nuclear import of classical NLS-bearing proteins is mediated by specific soluble factors, including Importin (Imp), which consists of two subunits, Impa and Impb, small GTPase Ran/TC4, and nuclear transport factor 2 [1]. The ternary complex with NLS-bearing protein, Impa, and Impb translocates into the nucleus, and the binding GTP-bound form of Ran to Impb triggers the dissociation of ternary complex, releasing Impa [2]. There are many additional pathways that mediate nuclear import; for example, Impb-like molecules (such as the transport factor for substrates carrying the M9 shuttling signal or importin 7) and Impb itself are competent to transfer some cargo by themselves [3]. It was previously reported that Impa could migrate into the nucleus in an Impb- and Ran-independent manner [4].
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