Placement of Leucine Zipper Motifs at the Carboxyl Terminus of HIV-1 Protease Significantly Reduces Virion Production

Pan, Yen-Yu; Wang, Shiu-Mei; Huang, Kuo-Jung; Chiang, Chien-Cheng; Wang, Chin-Tien

Culture media from transfected 293T cells were filtered through 0.45 mm-pore-size filters, followed by centrifugation through 2 ml of 20% sucrose in TSE (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA) plus 0.1 mM phenylmethylsulfonyl fluoride [PMSF]) at 4uC for 40 min at 274,0006g (SW41 rotor at 40,000 rpm). Viral pellets then were suspended in IPB (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 0.02% sodium azide) plus 0.1 mM PMSF. Cells were rinsed with ice-cold PBS (phosphate-buffered saline), scraped from the plates, collected in 1 ml of PBS and pelleted at 2,500 rpm for 5 min. The cell pellets were resuspended in 250 ml of IPB plus 0.1 mM PMSF, and then subjected to microcentrifugation at 4uC for 15 min at 13,7006g (14,000 r.p.m.) to remove cell debris. Either supernatant or cell sample was then mixed with equal volumes of 26 sample buffer (12.5 mM Tris-HCl pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue) and 5% b-mercaptoethanol and boiled for 5 min. Samples were subjected to SDS-PAGE and electroblotted onto nitrocellulose membranes. Membrane-bound Gag proteins were immunodetected using an anti-p24 gag (mouse hybridoma clone 183-H12-5C) monoclonal antibody at a 1:5,000 dilution from ascites. The secondary antibody was a rabbit anti-mouse (HRP)-conjugated antibody at 1:15,000 dilution as appropriate and the procedures used for HRP activity detection followed the manufacturer's protocol (Pierce). Immunodetected bands on film were quantified by using AlphaImager 2000 (Alpha Innotech Corp.) and Image J software.