GoKawiilCells
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 × 106 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. Resting purified CD4 T cells were cultured in presence of 10 IU ml−1 IL-2 without CD3/CD28 activation. Jurkat T cell lines clone E6-1 (ATCC TIB-152) and clone 1G5 (containing Tat-driven luciferase reporter, obtained from AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were cultured in complete RPMI medium. HEK293T/17 cells (ATCC, CRL-11268) and HeLa-TZMbl cells (CFAR) were cultured in DMEM GlutaMax medium (Thermo Fisher Scientific) supplemented with 10% FCS and 1% penicillin–streptomycin.
Plasmids
pNL4.3 plasmid (donated by M. Martin) was from the CFAR. pNL4.3 ΔEnv and pNL4.3 ΔVpr was provided by R. Sloan52. pCH040 and pCH077 were provided by G. Towers and were originally obtained through the NIH AIDS Reagent Program from J. Kappes and C. Ochsenbauer. pNLENG1-IRES NL4.3 Env and pNLENG1-IRES YU2 Env were provided by D. Levy53. pHGFP-GFPCA was generated previously and provided by V. Pathak and encodes GFP fused to CA in the NL4.3 ΔEnv backbone20. Parental pHGFP plasmid contains WT CA sequence20. pHGFP-mStayGold-CA was generated by replacing GFP sequence from pHGFP-GFPCA plasmid between the MluI and XbaI restriction sites with the mStayGold sequence cloned from the pRSETB/mStayGold plasmid (Addgene, 212017) using the following primers 5′-TCAGTGACGCGTATGGTGTCTACAGGCGAGGAGC and 5′-TCAGTCTCTAGACAGGTGGGCCTCCAGGGTC. pHGFP-Spot-tag-CA was generated by replacing the GFP sequence from the pHGFP-GFPCA plasmid between the MluI and XbaI restriction sites with the following primers also digested with MluI and XbaI enzymes: 5′-CGCGTCCAGACCGCGTGCGCGCCGTGAGCCATTGGAGCAGCGGCGGAT and 5′-CTAGATCCGCCGCTGCTCCAATGGCTCACGGCGCGCACGCGGTCTGGA. pBLAM-Vpr was obtained from Addgene and pAdVantage from Promega. pMDG expressing VSV glycoprotein was from Genscript. pWEAU_d15_410_5017 expressing HIV-1 WEAU isolate Env and pFurin were provided by L. McCoy. pCAT001 expressing codon-optimized HIV-1 HXB2 Env was obtained from Addgene (171061) NL4.3 Env-F522Y described previously17 was generated by site-directed mutagenesis using the QuickChange Lightning kit (Agilent) and the following primers: F522Y forward: 5′-GCTTTGTTCCTTGGGTAT TTGGGAGCAGCAGG; and F522Y reverse: 5′-CCTGCTGCTCCCAAA TACCCAAGGAACAAAGC.
Viruses
Viruses were made by transfecting 293T cells (7 × 106 cells seeded per T175 flask) with 10 μg HIV-1 plasmid using Fugene6 (Promega) in 1:3 ratio. HIV-1 NL4.3 ΔEnv and Env-F522Y virions were pseudotyped with Env from the primary isolate transmitter/founder virus WEAU by co-transfecting with pWEAU Env (5 μg) and pFurin (2.5 μg) plasmids. VSVg-pseudotyped NL4.3 ΔEnv and Env-F522Y were co-transfected with pMDG (2.5 μg). Blam-Vpr virus was made by co-transfecting pNL4.3 ΔVpr (10 μg), pBLAM-Vpr (2.5 μg) and pAdVantage (2 μg). GFP–CA virus was made by co-transfecting pNL4.3 WT (10 μg) with pHGFP-GFPCA (1 μg) as described previously20. mStayGold-CA virus was made by co-transfecting pHGFP (NL4.3 ΔEnv, 12 μg) with pHGFP-mStayGold-CA (1 μg) and pCAT001 HXB2 Env (1 μg) and pFurin (2.5 μg). Spot-CA virus was made by co-transfecting pHGFP (NL4.3 ΔEnv, 12 μg) with pHGFP-Spot-tag-CA (1 μg) and pCAT001 HXB2 Env (1 μg) and pFurin (2.5 μg). Viral supernatants were collected 48 h and 72 h after transfection, filtered through 0.45-μm syringe-driven filter, purified and concentrated by ultracentrifugation through 20% sucrose cushion and resuspended in complete RPMI medium. For experiments involving quantitative PCR (qPCR) analysis of viral DNA, the product supernatants were treated with DNase I (Sigma-Aldrich) for 1 h at 37 °C before ultracentrifugation. Viral titres were measured by quantifying supernatant RT activity by SG-PERT assay54. Alternatively, and where indicated, quantification of infectivity (TCID 50 per ml) was determined by titration of viral supernatants on HeLa-TZMbl reporter cells using Luciferase Bright-Glo substrate (Promega) and GloMax luminometer (Promega).
HIV-1 infections, VS priming and bead priming
Donor cell infection
Purified primary activated CD4 T cells (donors) were infected on day 5 after activation by spinoculation (2 h, 1,200g, 25 °C) with Env-pseudotyped NL4.3 Env-F522Y virus or NL4.3 ΔEnv virus (1.0 and 0.5 U RT per 106 cells, respectively) in the presence of DEAE-dextran (0.1 μg per 106 cells). Jurkat donor cells were infected with VSV-g pseudotyped NL4.3 Env-F522Y and NL4.3 ΔEnv virus (0.5U RT per 106 cells) by gravity infection for 4 h before changing the medium. Infected donor cells were incubated for 2 days before use, and infection was confirmed by flow cytometry.
VS priming
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