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.

In this study, we identify annexin II as a novel macrophage membrane-binding protein for the innate host defense protein, SLPI, by multiple parameters including im-munoprecipitation, mass spectrometry, peptide sequencing, and binding specificity. Striking was the ability of inhibitors of annexin II to mimic the kinetics and apparent mechanism of HIV-1 suppression by SLPI, denoting a shared site of action. Although not ruling out additional binding targets for SLPI, annexin II appears to be significant in mediating its anti-HIV-1 activity. Subsequent to binding of HIV-1 to the canonical receptors, CD4 and CCR5, HIV-1 fuses with the host cell membrane that might be facilitated by viral envelope PS (14) . PS is not encoded by HIV-1, but rather is acquired from its host cell membrane as it exits the cells (26) . During viral assembly at the cell surface or within cytoplasmic vesicles, cell membrane components become incorporated into the new viral coat along with virally encoded gp120/gp41. The resulting mosaic HIV-1 envelope represents a lipid bilayer with a unique cholesterol/phospholipid composition, embracing viral and host molecules, including PS. Although PS enhances infection, it does not mediate initial binding of the virus to the target cells (14), Figure 5 . Virus binding to annexin II. (A) Nunc immunoplates were coated with the indicated proteins overnight at 4ЊC, washed, and 100 l HIV-1 (10 4 /ml) was added for 1 h at 4ЊC. After six PBS washes, adherent virions were lysed and p24 was measured. Data represent mean and SEM (n ϭ 2). (B) HIV-1 was preincubated with excess soluble annexin II tetramer for 1 h before addition to macrophage cultures. After 2 h of coculture, nonadherent virus was removed, macrophages were cultured, and day 10 supernatants were assayed for p24 levels. (C) After treatment with anti-annexin II (␣Ann II), IgG, or SLPI for 30 min, macrophages were washed and HIV BaL was added for 30 min at 37ЊC. The cells were immediately washed and lysed, and bound p24 was assayed by ELISA. (D) Confocal microscope images (mid-cell single xy sections) of macrophages and apoptotic Jurkat cells, fixed 10 min into the process of phagocytosis. Jurkat cells were stained with the viable dye carboxyfluorescein diacetate succinimidyl ester (green) and macrophages were incubated with mouse anti-annexin II followed by Alexa 647-conjugated donkey anti-mouse secondary antibody (red), demonstrating annexin II staining within the phagosome membrane (arrows). (E) Nested PCR detecting proviral DNA on days 2 and 3 after HIV-1 infection in macrophages treated with DMEM (HIV), antiannexin II, or isotype control IgG. Control represents uninfected cells. consistent with our observations that neither soluble annexin II, RNAi, anti-annexin II, nor the annexin II ligand, SLPI, blocks HIV-1 binding, but rather inhibit postbinding and pre-reverse transcription, a point in the viral life cycle consistent with a proposed role for annexin II as a cellular fusogenic cofactor.