Abstract
BackgroundMyeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H2O2 and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected in vivo.FindingsThis report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV) glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner.ConclusionsOur data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide.
Highlights
Myeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H2O2 and chloride, which is released into body fluids
Generation of HOCl-modified serum albumins In a first experiment we tested different samples of the HOCl-modified HSA, which were generated by using different HOCl concentrations, in a syncytium inhibition assay
Binding of gp120 to HOCl-modified serum albumins Since a 1000-fold molar HOCl excess was sufficient to transform HSA into a viral entry inhibitor we generated HOCl-modified samples of three serum albumins from human, mouse and bovine by using a 1000-fold excess of HOCl. Using these three serum albumin preparations, we studied binding of modified human serum albumin (mHSA), mBSA and mMSA to LAV (NL4-3) gp120 by surface plasmon resonance spectroscopy (SPR) (Figure 2a, c, e)
Summary
Myeloperoxidase (MPO), an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl) from H2O2 and chloride, which is released into body fluids. In 1992, Klebanoff and coworkers [11] showed that stimulated polymorphonuclear (PMN) cells released an unknown factor which neutralized HIV-1. PMN from patients with hereditary myeloperoxidase (MPO) deficiency indicated that the antiviral activity was correlated with the presence and the release of the enzyme into cell culture medium. In addition to MPO, the presence of two other factors, H2O2 and chloride, was absolutely necessary to observe the antiviral activity in cell culture supernatants of stimulated PMN cells. Since the enzyme MPO catalyzes the reaction between H2O2 and chloride to generate HOCl (bleach), Klebanoff and coworkers [12] suggested that this product of the MPO/H2O2/halide system was directly responsible for HIV inactivation. A clue as to the substrate of the MPO/H2O2/halide system was provided by the detection of HOCl-modified proteins in human tissue by a specific monoclonal antibody (clone 2D10G9)
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