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Background:  HIV-1-infected monocyte-derived macrophages (MDM) are necessary for HIV-1 transmission, replication, and pathogenesis; however, no cellular proteins have been discovered that govern assembly and budding into internal vesicles. Previously, we discovered that HIV-1 p55^Gag binds to Annexin 2 (Anx 2), an endosome-associated protein that is involved in membrane organization and traffic and that cycles between the cytosol, the inner membrane of vesicles, and the plasma membrane. Our hypothesis is that the p55^Gag and Anx 2 interaction is involved in viral assembly/budding in MDM.

Methods:  MDM were treated with Anx 2 or scrambled Anx 2 siRNA. After 3 days, MDM were infected with 50 ng of HIV-1_JAGO for 16 hours, washed, and cultured for 4 days. Cell-culture supernatants were assayed for the presence of p24^Gag and duplicate cell cultures lysed for Anx 2 and GAPDH Western blotting or prepared for transmission electron microscopy as follows:  cultures were fixed for 1 hour in 2% glutaraldehyde, then scraped off the plate in 1% gelatin, and pelleted. The resulting cell pellet was fixed in 2% osmium, washed, dehydrated in ethanol and propylene oxide, embedded in plastic resin (EPON), 70-nm-thick sections cut, stained with uranyl acetate, and observed using a JEOL JEM 1010 transmission electron microscope.

Results:  Depleting Anx 2 protein with siRNA in MDM led to a significant decrease in p24^Gag and infectious virus release. Additionally, we observed no assembly or budding of HIV particles into internal vesicles (e.g., multivesicular bodies). Instead, we observed possible immature viral particles with membrane bilayer structures. Conversely, HIV infection of MDM expressing wild type levels of Anx2 (scrambled siRNA) resulted in HIV budding into internal vesicles and full particle maturation. We also determined that while Anx 2-depleted MDM released less infectious virus and p24^Gag protein, there was no change in the p24^Gag/infectious virus ratio.

Conclusions:  The reduction of Anx 2 protein inhibited HIV maturation in internal vesicles in MDM, which supports our hypothesis that binding of p55^Gag to Anx 2 is involved in directing viral assembly and budding to the endosomal membranes in MDM, and that reduction of Anx 2 leads to decreased production of mature HIV particles because this novel HIV maturation pathway has been disrupted.

Keywords: Gag; Assembly; Budding