Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

Ma, Ge; Greenwell-Wild, Teresa; Lei, Kejian; Jin, Wenwen; Swisher, Jennifer; Hardegen, Neil; Wild, Carl T.; Wahl, Sharon M.

PCR for Viral DNA. For a PCR-based assay for newly synthesized viral DNA (3), 200 l of DNase-treated HIV-1 BaL (10 4 / ml) was added to macrophages preincubated with anti-annexin II or control antibody for 30-60 min at 37 Њ C. After 2 h of coculture, the cells were washed three times with PBS, treated with trypsin-EDTA (0.05% trypsin and 0.53 mmol/l EDTA for 5 min at 24 Њ C) to remove noninternalized virus particles, washed twice with DMEM and 10% FCS, and incubated for 18-36 h. Cells were then washed, harvested in lysis buffer (100 mmol/l KCl, 10 mmol/l Tris HCl, pH 8.0, 2.5 mmol/l MgCl 2 , 0.5% Tween 20, and 0.5% NP-40), and 1 g of DNA was subjected to nested primer PCR amplification (25 l total volume). The first 35cycle round of amplification used primers corresponding to the env gene (nucleotide 8838 to 8358, HIV-1HXB2 sequence) and the U3 region of the 3 Ј long terminal repeat (nucleotide 9533 to 9558). Each cycle consisted of denaturation for 1 min at 94 Њ C, annealing for 1 min at 55 Њ C, and elongation for 1 min at 72 Њ C. PCR reactions (2.5 l each) from the first amplification were then subjected to a second 30-cycle amplification round using primers (5 Ј primer, nucleotide 8754 to 8782; 3 Ј primer, nucleotide 9436 to 9457) located within the nef gene. PCR products ( ‫ف‬ 730 bp) from the second amplification were visualized by ethidium bromide staining after agarose gel electrophoresis.