Elisa for p24

HIV-1 capsid cores were prepared using a protocol based on Shah and Aiken (2011 (link)). 90 ml HEK293T supernatant containing VSV-G pseudotyped HIV-1 GFP was pelleted over 20% sucrose dissolved in core prep buffer (CPB; 20 mM Tris (pH 7.4), 20 mM NaCl, 1 mM MgCl2) in an SW28 rotor (Beckman) at 25,000 rpm at 4°C. Pellets were gently resuspended at 4°C in CPB for 1 hr with occasional agitation. Resuspended pellets were treated with DNase I from bovine pancreas (Sigma Aldrich) for 1 hr at 200 µg/ml at room temperature to remove contaminating extra-viral DNA. Virus was subjected to spin-through detergent stripping of the viral membrane as follows. A gradient at 80–30% sucrose was prepared in SW40Ti ultracentrifuge tubes and overlaid with 250 µl 1% Triton X-100 in 15% sucrose, followed by 250 µl 7.5% sucrose. All solutions were prepared in CPB. 750 µl DNase-treated, concentrated virus was layered on top of the gradient and subjected to 32,500 rpm at 4°C for 16 hr. The preparation was fractionated and the location of cores was determined by ELISA for p24 (Perkin Elmer). Core-containing fractions were pooled and snap frozen before storage at −80°C.

One × 10⁶ 293 T cells were seeded into 2 × 10 cm dishes in inositol-free DMEM and left to adhere overnight. The media was replaced with 5 ml inositol-free DMEM supplemented with 5 µCi/ml ³H-inositol (Perkin Elmer). After 3 days incubation, an additional 5 ml inositol-free media containing 5 µCi/ml ³H-inositol was added onto cells, which were then transfected with pMDG2, pCRV GagPol and CSGW. Cells were left for a further 3 days to produce VSV-G pseudotyped HIV1. Viral supernatants were topped up to 30 ml and pelleted over a 5 ml 20% sucrose cushion) in a SW28 rotor (Beckman) at 28,000 rpm at 4°C. Pellets were resuspended in inositol-free media and pelleted as previously. After the second spin, pellets were resuspended in 1 ml PBS and spun at 13,000 rpm at 4°C in a bench top microfuge for 60 min. Pellets were frozen at −20˚C until processing. Cells were washed with PBS, then harvested by scraping, counted and pelleted for quantification of cellular IP₆ labelling. Pellets were frozen at −20˚C until processing. For comparison of virion and purified capsid core samples, p24 levels were determined by ELISA for p24 (Perkin Elmer).