Abstract
Microvirin (MVN), a recently isolated lectin from the cyanobacterium Microcystis aeruginosa PCC7806, shares 33% identity with the potent anti-human immunodeficiency virus (HIV) protein cyanovirin-N (CV-N) isolated from Nostoc ellipsosporum, and both lectins bind to similar carbohydrate structures. MVN is able to inhibit infection by a wide variety of HIV-1 laboratory-adapted strains and clinical isolates of different tropisms and subtypes in peripheral blood mononuclear cells. MVN also inhibits syncytium formation between persistently HIV-1-infected T cells and uninfected CD4(+) T cells and inhibits DC-SIGN-mediated HIV-1 binding and transmission to CD4(+) T cells. Long term passaging of HIV-1 exposed to dose-escalating concentrations of MVN resulted in the selection of a mutant virus with four deleted high mannose-type glycans in the envelope gp120. The MVN-resistant virus was still highly sensitive to various other carbohydrate binding lectins (e.g. CV-N, HHA, GNA, and UDA) but not anymore to the carbohydrate-specific 2G12 monoclonal antibody. Importantly, MVN is more than 50-fold less cytotoxic than CV-N. Also in sharp contrast to CV-N, MVN did not increase the level of the activation markers CD25, CD69, and HLA-DR in CD4(+) T lymphocytes, and subsequently, MVN did not enhance viral replication in pretreated peripheral blood mononuclear cells. Therefore, MVN may qualify as a useful lectin for potential microbicidal use based on its broad and potent antiviral activity and virtual lack of any stimulatory properties and cellular toxicity.
Highlights
Already caused an estimated 25 million deaths worldwide
Carbohydrate binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), Urtica dioica agglutinin (UDA) and BanLec, or the prokaryotic cyanovirin-N (CV-N) and griffithsin bind to multiple glycans that are present on the envelope of human immunodeficiency virus (HIV) and subsequently inhibit the viral entry process (4 – 8)
MVN was evaluated in peripheral blood mononuclear cells (PBMCs) against a wide variety of human immunodeficiency virus type 1 (HIV-1) clades and showed antiviral activity against all HIV-1 group M clades with IC50 values ranging between 2.1 and 167 nM
Summary
Test Compounds and Monoclonal Antibodies—The mannose-specific lectin MVN (14.3 kDa) from the microcystin-producing strain M. aeruginosa PCC7806 was expressed in Escherichia coli and purified as described previously [24]. Human T-lymphocytic C8166, HUT-78, and SupT1 cells were obtained from the American Type Culture Collection (Manassas, VA) These cell lines were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (BioWittaker Europe, Verviers, Belgium) and 2 mM L-glutamine (Invitrogen) and maintained at 37 °C in a humidified CO2-controlled atmosphere. Antiviral Testing of MVN against HIV-1 Isolates in PBMCs— PHA-stimulated blasts were seeded at 0.5 ϫ 106 cells/well into a 48-well plate containing varying concentrations of compound in medium containing IL-2. The cells were collected, washed in culture medium, suspended in RPMI medium with 2 ng/ml IL-2, and seeded in a 48-well flat bottom plate (5 ϫ 105 cells in 450 l), and 50 l of the CCR5-tropic clade B HIV-1 BaL stock was added at ϳ100 TCID50. Statistical analysis was performed with GraphPad Prism 5 statistical software (GraphPad Software Inc., San Diego, CA)
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