Role of nucleoporins in nuclear import and 3D location of HIV-1 in the nuclear compartment

Abstract

BackgroundEmerging evidence suggests a fundamental role of nucleo-porins (Nups) during HIV-1 replication. The involvementof Nups in HIV-1 life cycle was demonstrated boththrough genome-wide siRNA screens and detailed mole-cular analyses. Nevertheless, the interaction of HIV-1 withNups has been poorly described: the impact of Nupsduring viral import through the nuclear pore complex(NPC) and during subsequent events within the nuclearcompartment still remains unclear. The aim of this studyis to identify the role of specific Nups in the nuclearimport of HIV pre-integration complexes (PICs) andduring downstream events leading to integration inspecific regions of the cellular chromatin.Materials and methodsThe nuclear import of the PICs was evaluated in Nupdepleted cells using an imaging technique which providesadirectvisualizationofviruses inside the cells. Knock-down cells were generated though silencing technology(siRNA and shRNA). After silencing, the cells wereinfected with HIV-IN-EGFP viruses carrying integrasefused to EGFP [1]. The comparative analysis of intra-nuclear fluorescent viruses in Nup depleted cells allowedus to determine the implication of Nups in HIV-1 nuclearimport.ResultsHere we demonstrate that selective depletion of nucleo-porins 153 and 98, which led to considerable reduction inHIV-1 infectivity, determines an impairment in nuclearPICs trafficking though the NPCs. Besides controllingnucleo-cytoplasmic exchange, several Nups might dynami-cally interact with PICs away from the NPC. The interac-tion between HIV and Nups may regulate the HIV-1events downstream nuclear import. Therefore, weexploited the single-cell imaging of HIV-1 provirus (SCIP)technique, recently set up in our lab [2], to determine theputative role of Nups in integration by 3D analysis ofthese spots.ConclusionsHIV visualization technique used in this study provides adirect measurement of the nuclear import of PICs asopposed to indirect 2-LTR circles measurement. The useof this direct assay coupled with SCIP technique mighthelp us to improve our knowledge on the involvement ofNups in the nuclear import of HIV and in subsequentevents within the nuclear compartment.