Our study set out to identify the implication of nucleoporins, previously found to be involved in HIV-1 infectivity and/or nuclear import, in the actual translocation process through the NPC. We found that although all studied nucleoporins impaired HIV-1 infection upon depletion, only two (Nup358/RanBP2, and Nup153) were involved in nuclear import and indeed only one (Nup153) showed any evidence of actual participation in translocation through the NPC. This observation emphasises that the manipulation of NPCs by viruses is complex and not limited to mere translocation through the nuclear pore. Viruses may usurp cellular nucleoporins for docking 
, chromosomal site selection for integration 
, and disruption of nucleo-cytoplasmic trafficking 
We previously hypothesised that HIV-1 capsids might dock directly on cytoplasmic filaments of the nuclear pore, based on the confined vibratory movement of HIV-1 complexes docked at the nuclear membrane 
, and the localisation of capsids at the nuclear pore frequently off-centered relative to the lumen of the pore 
. Here we show that HIV-1 capsid cores bind to Nup358/RanBP2, thought to be the main component of NPC cytoplasmic filaments 
. Interestingly, NPCs lacking Nup358/RanBP2 and devoid of cytoplasmic filaments were shown to maintain importin alpha/beta- and transportin-dependent import 
, which emphasises that depletion of Nup358/RanBP2 specifically impairs HIV-1 docking at the NPC rather than disrupting importin−/transportin-mediated nuclear import of HIV-1. Previous evidence suggests that Nup214/CAN mediates NPC docking of adenoviruses 
and of herpes virus together with importin ß and Nup358/RanBP2 
. In the case of HIV-1, data here show that Nup214/CAN is not involved in viral docking or nuclear import but that its apparent effect on HIV-1 infectivity is limited to a non-specific inhibition of mRNA export.
After entry in target cells, HIV-1 sheds its capsid shell during a step referred to as uncoating. However, where and when this occurs and the nature of the trigger (cellular or viral) remain a matter of debate. Reports of cytoplasmic HIV-1 complexes of broadly varying sizes and shapes suggest that uncoating could occur gradually during transport to the nuclear envelope 
, presumably in response to successive changes in the cellular environment. However, increasing data supports the presence of HIV-1 capsid at the nuclear pore and/or acting as a determinant of HIV-1 nuclear import 
. Uncoating certainly also occurs during cytoplasmic transport, and possibly accounts for the majority of incoming viral complexes. However, these may correspond to viral complexes destined for or undergoing degradation, for instance following entry by endocytosis 
. Our work shows that HIV-1 capsid interacts with Nup358/RanBP2 () and that depletion of Nup358/RanBP2 impairs arrival of HIV-1 complexes at the nuclear envelope, thus confirming the presence of HIV-1 capsid cores at the nuclear membrane (). However, our study does not show any effect for Nup358 in integration, since the strong reduction in proviral integration is simply due to a strong nuclear import defect () and considering the exclusive cytoplasmic location of Nup358/RanBP2 we do not expect to find its potential viral partner in the nucleus. We cannot exclude that the absence of this nucleoporin could affect HIV-1 site integration 
but that may reflect the change of chromatin environment associated with depletion of Nup358 
. In this work, we identified Nup358/RanBP2 as docking factor for HIV-1 capsid using independent techniques to assess docking (microscopy, cell fractionation) and interaction (in vitro
CA-NC binding). We are the first to identify the binding of Nup358/RanBP2 to HIV-1 in vitro
assembled CA-NC complexes suggesting that this interaction is important for HIV-1 nuclear import.
Nup358/RanBP2 is one of over 20 human proteins that contain a cyclophilin-like domain 
. The high homology between the human Nup358/RanBP2 cyclophilin-like domain and human CypA likely accounts for its ability to mediate docking of HIV-1 capsids at the nuclear pore. Indeed, a direct interaction between the Nup358/RanBP2 cyclophilin domain and HIV-1 CA N-terminal domain (NTD) was recently shown by calorimetric assay 
. In this study, we demonstrate binding between in vitro assembled HIV-1 CA-NC and full-length Nup358/RanBP2, and show ~3-fold reduced binding for GFP-RanBP2-ΔCyp deletion mutant when compared to GFP-RanBP2, suggesting the importance of this region that contains a cyclophilin-homology domain to have an efficient docking step of HIV at the NPC. Although we cannot exclude that a misfolding of the ΔCyp mutant accounts for reduced binding to HIV-1 cores, the mutant localised correctly at the nuclear membrane and did not form aggregates that precipitate in the absence of capsid, suggesting that this is an unlikely explanation. Interestingly, ΔCyp mutant maintained residual ability to interact with HIV-1 cores suggesting that other Nup358/RanBP2 domains, may also contribute to this binding. FG domains localised throughout the protein could represent the other part of the Nup358/RanBP2 with residual binding activity for HIV-1 CA-NC. Future experiments will be carried out to identify the amino acid residues of Nup358/RanBP2 essential for binding to HIV-1 cores.
In our study, Nup153, located in the nuclear basket of NPC, was the only nucleoporin involved in HIV-1 translocation through the nuclear pore, rather than in other NPC-related steps. Interestingly, it is the only nucleoporin that was identified in all four HIV genome-wide screens 
. We agree with other reports showing Nup153 involved in HIV-1 nuclear import 
. The position of Nup153 in the nuclear basket leads us to hypothesise that exit from the nuclear basket, rather than entry into the lumen of the pore, might be critical for passage of HIV-1 into the nucleus. Based on recent single cargo translocation imaging 
, initial translocation of HIV-1 may involve a series of low-affinity, non-covalent and reversible interactions with FG domains from multiple Nups and diffusion through the NPC, whereas interaction with Nup153 might be critical for mediating irreversible and directional exit from the NPC. Interaction with FG-repeats may be mediated indirectly by nuclear transport receptors, such as importin 7 
or transportin 3 
After exit from the nuclear basket, both Nup98 and Nup153, which shuttle on and off the NPC 
and have been shown to interact with chromatin 
, might accompany the PIC to its integration site. In particular, Nup98 has been found to localise to the nucleoplasm and participate in chromosomal remodelling and regulation of gene expression 
. It will be interesting to determine whether Nup98 and/or Nup153 are hijacked by HIV-1 for transport to euchromatin and contribute to specific site selection in expressed genes, since previous work has shown that HIV-1 integrates preferentially within actively transcribed genes 
. Surprisingly, Nup98 depletion affected HIV-1 and MLV infection equally, but the reduction of MLV infectivity could merely be due to the slight accumulation of cells in G1/S phase that we observed since MLV enters the nucleus during metaphase.
Our work demonstrates that a key to the ability of HIV-1 to replicate in non-dividing cells is its capacity to use NPC components for its active transport across the nuclear pore, thus underlining the evolutionary adaptability of HIV-1 to exploit host mechanisms to achieve active nuclear import. Our study suggests a new appealing role for the NPC in HIV-1 infection proposing that the viral nuclear entry step may be important not only for actual translocation, but also for correct subsequent integration as a result of the physical interaction that exists between nuclear pore baskets and the chromatin (). The study of the physical and functional interactions between HIV-1 and the NPC not only contributes to our understanding of how other viruses manipulate the nuclear pore but also strengthen our comprehension of lentiviral vectors used for gene transfer protocols, whose active nuclear import is similar to that of HIV-1.
Overview of the functional interactions between nucleoporins and HIV-1 during infection.