Docking of HIV-1 Vpr to the Nuclear Envelope Is Mediated by the Interaction with the Nucleoporin hCG1

Abstract

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast twohybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.In contrast to oncoretroviruses that replicate only in dividing cells and enter the nucleus upon nuclear breakdown during mitosis, HIV-1 1 and other lentiviruses have the ability to infect non-dividing cells, such as macrophages and quiescent T lymphocytes. After entry of the virus into the cell, the HIV capsid seems to uncoat rapidly. The genomic HIV-1 RNA is reverse transcribed into linear double-stranded DNA, which remains associated with a nucleoprotein complex, called the preintegration complex (PIC). The viral DNA is then imported into the nucleus through the nuclear envelope (NE) via an active mechanism within 4 -6 h after infection (1).In eukaryotic cells, the NE creates distinct nuclear and cytoplasmic compartments. This structure consists of two concentric membranes, the inner and outer nuclear membranes which are continuous with the endoplasmic reticulum. The NE is stabilized by the nuclear lamina, a tight meshwork of intermediate filament proteins underlying the inner nuclear membrane (for review, see Ref.2), whereas on the outer side, cytoplasmic intermediate filaments in close contact with the nucleus serve to suspend the nucleus in the cytoplasm (3). Spanning both membranes are nuclear pore complexes (NPC) that form aqueous channels, which allow selective traffic between nucleus and cytoplasm and impose a permeability barrier to free diffusion of macromolecules or complexes. The NPC is a large supramolecular structure (4) formed of ϳ30 unique proteins in vertebrates, termed nucleoporins (Nups) giving rise to an estimated molecular mass of 60 MDa (Ref. 5; for reviews, see Refs. 6 and 7). High resolution electron microscopic images of NPCs reveal an 8-fold symmetric structure, formed by nuclear and cytoplasmic rings and a central spoke complex. Peripheral filaments emanate from the core of the complex into the nucle...

Vpr-mediated Incorporation of UNG2 into HIV-1 Particles Is Required to Modulate the Virus Mutation Rate and for Replication in Macrophages

“…The concentration of the fusion proteins was estimated on a SDS-PAGE gel stained with Coomassie Blue G-250 (Bio-Rad) and determined relative to a range of bovine serum albumin standard. Cell lysates from 6 ϫ 10 6 HeLa cells expressing HA-Vpr (WT or W54R) were prepared and incubated with purified GST or GST-UNG proteins as previously described (4). Bound proteins were then resolved by SDS-PAGE and then analyzed by Western blotting as described (4).…”

“…Cell lysates from 6 ϫ 10 6 HeLa cells expressing HA-Vpr (WT or W54R) were prepared and incubated with purified GST or GST-UNG proteins as previously described (4). Bound proteins were then resolved by SDS-PAGE and then analyzed by Western blotting as described (4).…”

“…Human immunodeficiency virus type 1 (HIV-1) 1 Vpr is a 96-amino acid non-structural protein that is associated with virus particles and can accumulate at the nuclear envelope and in the nucleus of infected cells (1)(2)(3)(4). The incorporation of Vpr into particles requires a direct interaction with the p6 region of the Gag polyprotein precursor (5,6).…”

“…Finally, Vpr is required for the infection of nondividing cells, and this requirement is related, at least in part, to its role in the nuclear translocation of the preintegration complex (PIC) containing the viral DNA. Vpr possesses an affinity for the components of the nuclear pore complex, and it has been proposed that Vpr may facilitate the nuclear translocation of the PIC across the nuclear envelope (4,(13)(14)(15)(16). Infection of nondividing, terminally differentiated macrophages and resting T cells represents a viral reservoir in the host that is crucial for subsequent virus spread to lymphoid organs and T-helper lymphocytes and finally for AIDS pathogenesis (17).…”

Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions

“…Experiments in macrophages strongly suggest an important role for Vpr in mediating the nuclear import of HIV-1 PICs into the nucleus of nondividing cells [20]. The mechanism of Vpr-mediated nuclear import is not clear, it is likely that Vpr interacts directly or indirectly with cellular machinery regulating the nucleo-cytoplasmic shuttling [21][22][23][24][25].…”