Simian Immunodeficiency Virus Vpx Is Imported into the Nucleus via Importin Alpha-Dependent and -Independent Pathways

Abstract

Vpx protein of human immunodeficiency virus type 2/simian immunodeficiency virus (SIV) has been implicated in the transport of the viral genome into the nuclei of nondividing cells. The mechanism by which Vpx enters the nucleus remains unknown. Here we have identified two distinct noncanonical nuclear localization signals (NLSs) in Vpx of SIV smPbj1.9 and defined the pathways for its nuclear import. Although nuclear targeting signals identified here are distinct from known nuclear import signals, translocation of Vpx into the nucleus involves the interaction of its N-terminal NLS (amino acids 20 to 40) or C-terminal NLS (amino acids 65 to 75) with importin alpha and, in the latter case, also with importin beta. Collectively, these results suggest that importins interact with Vpx and ensure the effective import of Vpx into the nucleus to support virus replication in nondividing cells.Primate lentiviruses are able to infect nondividing cells, such as terminally differentiated macrophages (4,12,15,16,19), a feature that distinguishes them from the onco-retroviruses, which require nuclear membrane dissolution during cell division for successful viral replication (7,31,44). Vpx of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) is a 112-amino-acid (aa), 18-kDa protein (20). It is expressed in infected cells in a Rev-dependent manner and is packaged into new virions through its interaction with the p6 region of the p55 gag precursor (1,8,20,26). As the Vpx protein is incorporated into the virion (27,28,32), it becomes available during early replication events, immediately following entry of the new virion into a target cell even before de novo viral protein synthesis can start (10,21,32,37,38). Based on such late expression during virus production and early availability during initial infection, it has been proposed that Vpx is involved in the early stages of the viral life cycle, particularly in the efficient import of the viral genome into the nuclear compartment of nonproliferating target cells (10,32,37,39,40,42). Vpx was found to participate in the active translocation of the large (Stokes radius, 28 nm) viral preintegration complex (PIC) across the limiting nuclear pore (10). Although Vpx displays evident karyophilic properties, it does not contain a canonical nuclear localization signal (NLS) (32,37,39,40). The mechanism of Vpx-mediated nuclear import appears novel, but how Vpx cooperates with viral and host proteins to transport the viral PIC into the nucleus remains elusive.Eukaryotic cells possess double-layered nuclear membranes containing multiple nuclear pores that regulate bidirectional transport of macromolecules that are critically required for the maintenance of normal cellular physiology. The nuclear pore complex (NPC) spans the nuclear membrane and creates an aqueous channel with a pore diameter of 9 nm, allowing the theoretical passive diffusion of a protein up to approximately 30 to 40 kDa (5,9,17,18,35). Translocation across the NPC and into the nucleoplasm and...

Nuclear Export of Simian Immunodeficiency Virus Vpx Protein

“…GFP fusions containing different Vpx deletion fragments expressed in either HeLa or Cos-7 cells were found to localize differentially (GFPVpx1-63 in the cytoplasm and GFP-Vpx20-40 in the nucleus) (48). This indicated the presence of NES in SIVsm Vpx.…”

“…SH3 domains of Fyn, Src, Hck, full-length Crk, and importin-␤ as glutathione transferase (GST) fusion proteins were expressed in E. coli BL21(DE3) or M15-prap4 cells and purified as described previously (48). GFP and GFP-Vpx (full length) were synthesized using […”

Hsp40 Facilitates Nuclear Import of the Human Immunodeficiency Virus Type 2 Vpx-Mediated Preintegration Complex

“…Furthermore, we performed in vitro GST pull-down competition assays (data not shown). In these studies, GST-Gag was used to bind 35 Slabeled Vpx in the presence of increasing amounts of Histagged Hsp40/DnaJB6. The result demonstrated that a reduced amount of Vpx was captured by GST-Gag when Histagged Hsp40/DnaJB6 was preincubated with Vpx, suggesting that Hsp40/DnaJB6 competes with Gag for binding to Vpx.…”

“…Moreover, N-terminal (residues 20 to 40) and C-terminal residues (101 to 112) may also affect the nuclear import of Vpx (20,26,35). It has been suggested that importins ␣ and ␤ contribute to this activity (35). Although expression of Vpx, in the absence of other viral proteins, results in a predominant nuclear localization, coexpression of the Pr55 Gag precursor protein shifts Vpx to the plasma membrane and virus particles (26,27).…”

“…Vpx contains a nuclear localization signal (NLS) within amino acids 65 to 72 (3,20). Moreover, N-terminal (residues 20 to 40) and C-terminal residues (101 to 112) may also affect the nuclear import of Vpx (20,26,35). It has been suggested that importins ␣ and ␤ contribute to this activity (35).…”