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.

HIV-1 Infection. Adherent macrophages were infected with R5 HIV-1 BAL grown in primary human macrophage cultures (50 l TCID 50 ϭ 10 4 /ml; Advanced Biotechnologies Inc.) for 2 h at 37 Њ C, unbound virus was washed away, and the cells were cultured in DMEM containing 10% FCS at 37 Њ C for 1-14 d (2, 3). One half of the supernatant was collected every 2-3 d and replaced with fresh medium. Supernatants were tested for HIV-1 p24 antigen by ELISA (PerkinElmer). For preparation of additional isolates, 60 ϫ 10 6 PHA-activated PBMCs were pelleted for 10 min at 1,500 rpm in a 15-ml conical tube, the supernatant was removed, and 1 ml of virus seed stock (HIV-1 JRCSF, ADA, and primary isolate Clade B 92US712; NIH AIDS Research and Reference Reagent Program) was added to the target cells with gentle mixing. After 1-2 h of incubation, the cell/virus mixture was diluted to 30 ml with complete media (RPMI 1640, 10% FCS, 1% gentamycin, and 5% IL-2) and transferred to a T-25 tissue culture flask. On days 2, 4, 6, and 8 after infection, the cells were pelleted, the media was replaced, and day 6 and 8 culture supernatants were tested for p24 and RT activity, filtered through a 0.45-mm filter, vialed as 1.0-ml aliquots, and stored at Ϫ 70 Њ C before infection of macrophages.