Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore, Nuclear Import and Integration

Francesca Di Nunzio,Nathalie J Arhel,Anne Danckaert,Patricio Perez,Felipe Diaz-Griffero,Juliette Fernandez,Thomas Fricke,Pascal Roux,Emmanuelle Perret,Spencer Shorte,Pierre Charneau

doi:10.1371/journal.pone.0046037

Abstract

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

Highlights

The nuclear pore complex (NPC) is a supramolecular protein assembly forming a highly selective channel embedded in the nuclear membrane
The ratio bound/input for GFP-RanBP2 and GFP-RanBP2-DCyp is 160.19 and 0.3960.2, respectively (Fig. 5C). These experiments showed that GFP-RanBP2-DCyp binds,3-fold less than wild type in vitro assembled Human Immunodeficiency Virus (HIV)-1 CANC complexes. These results suggested that the C-terminus region of Nup358/RanBP2 contributes to the ability of Nup358/RanBP2 to bind Human Immunodeficiency Virus type 1 (HIV-1) CA-NC, but that other parts of the protein, might contribute to guaranty an efficient binding between the cytoplasmic filaments of the NPC, RanBP2 and HIV-1 CA, supporting the notion that Nup358/RanBP2 is involved in the docking for HIV at the nuclear pore
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

Summary

Introduction

The nuclear pore complex (NPC) is a supramolecular protein assembly forming a highly selective channel embedded in the nuclear membrane. It regulates bidirectional nucleo-cytoplasmic transport for a large range of proteins and complexes too large to diffuse freely through the NPC [1], [2], [3]. They are composed of numerous copies of ,30 different nucleoporins (Nups), which have a well-assigned localisation, function and half-life, and are present as multiples of eight reflecting the highly conserved eight-fold axial symmetry of NPCs [2], [4], [5], [6]. On the nuclear side of the NPC, Nups such as Nup153 and Nup associate with the nuclear basket and with the chromatin both in proximity of and away from the NPC [9]

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Results

Conclusion