Isolation and Characterization of Niphatevirin, a Human‐Immunodeficiency‐Virus‐Inhibitory Glycoprotein from the Marine Sponge Niphates Erecta

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Abstract
Anti‐human immunodeficiency virus (HIV)‐bioassay‐guided fractionation of aqueous extracts of the Caribbean sponge Niphates erecta led to isolation of a novel anti‐HIV protein, named niphatevirin. The protein was purified to homogeneity by ethanol precipitation, ammonium sulfate precipitation, gel‐permeation chromatography and concanavalin‐A–Sepharose affinity chromatography. Niphatevirin potently inhibited the cytopathic effects of HIV‐1 infection in cultured human lymphoblastoid (CEM‐SS) cells; the effective concentration of drug that results in 50% protection of the cells through inhibition of cell lethality, cell‐cell fusion and syncytium formation was approximately 10 nM. Delay of addition of niphatevirin to infected cultures by two hours markedly decreased (≈50%) cytoprotection; delay of addition by eight hours resulted in no antiviral activity. Niphatevirin bound to CD4 in a manner that prevented the binding of gp120, but did not directly bind gp120. Niphatevirin (6.5 μM) was inactive in both hemagglutination and hemolysis assays. Niphatevirin had a molecular mass of about 19 kDa by matrix‐assisted laser‐desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry, and a native molecular mass of approximately 18 kDa by gel‐filtration chromatography. The protein had an acidic isoelectric point of 4.2–4.6, and was shown by periodate acid Schiff's staining to be glycosylated.