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.

Annexin II Is Associated with Entry/Fusion of HIV-1 in Macrophages. Next, we focused on when in the virus life cycle annexin II cooperates with HIV-1 to promote infection. Consistent with SLPI (2, 3), blockade of annexin II on macrophages did not significantly interrupt HIV-1 binding to the cells (Fig. 5 C) , likely dissociating it from a direct interaction with CD4 and/or CCR5. In confirmatory studies, anti-annexin II was incorporated into a fusion assay in which effector cells expressing recombinant Env, but lacking viral PS, were cocultured with target cells expressing recombinant CD4 and coreceptors (23) . In the absence of PS, anti-annexin II was ineffective in interrupting this fusion process (not depicted), ruling out a specific interaction with Env, CD4, and/or coreceptors. These data emphasize the potential unique constraints of macrophage-HIV-1 entry events that might be optimized through an annexin II-PS cofactor linkage. Although the role of annexin II in viral entry may involve participation in internalization or structural/functional facilitation of fusion events, it was unclear whether membrane annexin II had intracellular access. Because apoptotic cells also bind through PS to annexin II on macrophages (24, 25), we exposed macrophages to apoptotic Jurkat cells (PS ϩ ) as a model of annexin II-dependent internalization. By immunofluorescence, annexin II could be found within the early phagosome membrane, consistent with surface membrane internalization (Fig. 5 D) . Whether internalization is essential to viral entry is under investigation, but by multiple criteria, the role of annexin II appears instrumental early in the infection process. To confirm that disconnecting the HIV-annexin II bond inhibits the virus Figure 4 . Annexin II as a cofactor in HIV infection. (A) Adherent macrophages in 48-well plates were treated or not with 25 g/ml anti-annexin II for 60 min at 37ЊC before infection with M tropic HIV-1 BaL for 2 h, the unbound virus was washed away, and the cells were cultured in DMEM containing 10% FCS. Every 2-3 d, aliquots of supernatants were removed, replaced with fresh medium, and tested for HIV-1 p24 antigen. (B) Macrophages, treated or not with anti-annexin, IgG, or SLPI for 60 min, were infected with HIV-1 and 14-d supernatants were tested for p24. A representative experiment (n ϭ 4) is shown. (C) SiRNA sequences 1, 2, and control (cSi) were transfected into monocytes using the Amaxa Nucleofector kit, in parallel with an electroporation control (EP) and an untreated control (Ctrl). The cells were incubated 6 d before analysis of annexin protein by Western (inset) and infection with HIV-1. Supernatants were assayed for HIV-1 p24 10 d after infection (n ϭ 5). (D) Macrophages treated with siRNA as indicated in C were analyzed for annexin II, CD4, and CCR5 protein by Western blot. (E) Macrophages were treated with anti-annexin II, infected with HIV-1 JRCSF (JRC; TCID50 ϭ 250/ml), ADA (TCID50 ϭ 10,000/ml), or primary isolate Clade B 92US712 (TCID50 ϭ 1,000/ml), washed, and cultured, with supernatants collected for p24 ELISA every 2-3 d for 12 d. (F) PHA-blasted T cells were treated with anti-annexin II or isotype IgG before infection with T cell tropic HIV IIIB. P24 levels on day 7 are shown. life cycle after HIV-1 binding but before reverse transcription, as shown for SLPI (3), macrophages were infected with or without annexin II inhibitors and the formation of nascent viral DNA was assessed using a nested PCR