HIV-1 signalling remodels nuclear pores to licence infection

Cells Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte cones from healthy donors (NHS Blood and Transplant Services) by density centrifugation using Lympho 24+ Spin medium (PluriSelect) and pluriMate II tubes (PluriSelect). PBMCs were cryopreserved in 10% DMSO (Sigma-Aldrich) in FCS (LabTech). CD4 + T cells were isolated by negative selection using the MojoSort Human CD4 T cell Isolation Kit (BioLegend) and cultured in RPMI1460 medium supplemented with 10% FCS, 1% penicillin–streptomycin and 1% GlutaMax (Thermo Fisher Scientific) (complete RPMI medium) with 10 IU ml −1 IL-2 (Center for AIDS Reagents (CFAR), National Institutes of Biological Standard and Control, UK). For CD25/CD69 depletions biotin-Ab cocktail from the MojoSort Human CD4 T cell isolation kit was supplemented with biotin–anti-CD25 (M-A251, BioLegend) and biotin–anti-CD69 (FN50, BioLegend) antibodies (both at 10 μg per 50 million cells) to retain CD25/CD69 expressing cells in the negative fraction. Purity was assessed by flow cytometry (described below). Isolated CD4 T cells (8–12 × 10 6 cells) were activated in Nunclon Delta T25 flasks (Thermo Fisher Scientific) using plate-bound anti-CD3 antibody (OKT3, 5 μg per flask, BioLegend) and soluble anti-CD28 antibody (CD28.2, 2 μg ml −1 , BioLegend). After 3 days of activation, cells were cultured/rested in fresh medium (without CD3/CD28 antibodies) for another 2 days before being used for infections or co-cultures. …