ABSTRACT
Aqueous extracts of the New Zealand sponge Adocia sp. (Haplosclerida) displayed potent anticytopathic activity in CEM-SS cells infected with HIV-1. Protein fractions of the extract bound both to the viral coat protein gp120 and to the cellular receptor CD4, but not to other tested proteins. The purified active protein, named adociavirin, was characterized by isoelectric focusing, amino acid analysis, MALDI-TOF mass spectrometry and N-terminal sequencing. Adociavirin, a disulfide-linked homodimer with a native molecular weight of 37 kDa, was active against diverse strains and isolates of HIV-1, as well as HIV-2, with EC50 values ranging from 0.4 nM to > 400 nM. The anti-HIV potency of adociavirin appears dependent on host cell type, with macrophage cultures being the most sensitive and peripheral blood lymphocytes the most resistant.
Subject(s)
Anti-HIV Agents/isolation & purification , HIV-1/drug effects , Porifera/chemistry , Proteins/isolation & purification , Amino Acid Sequence , Animals , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacology , CD4 Antigens/metabolism , Cell Fusion/drug effects , Cell Line , Cytopathogenic Effect, Viral , HIV Envelope Protein gp120/metabolism , Molecular Sequence Data , Proteins/metabolism , Proteins/physiologyABSTRACT
Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.
Subject(s)
Amino Acid Sequence/physiology , Anti-HIV Agents/chemistry , Bacterial Proteins/chemistry , Carrier Proteins/chemistry , Amino Acid Sequence/genetics , Anti-HIV Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cysteine/genetics , Disulfides/chemistry , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Structure, Tertiary , Sequence Analysis , Sequence Deletion , Sequence Homology, Amino Acid , Serine/genetics , Structure-Activity RelationshipABSTRACT
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 (approximately 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 microM) 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.
Subject(s)
Anti-HIV Agents/isolation & purification , Carrier Proteins/isolation & purification , Glycoproteins/isolation & purification , HIV-1/drug effects , Plant Lectins , Porifera/chemistry , Agglutination Tests , Amino Acids/analysis , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Anti-HIV Agents/pharmacology , Antiviral Agents/chemistry , CD4 Antigens/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Carrier Proteins/pharmacology , Cell Line , Dithiothreitol/pharmacology , Edetic Acid/pharmacology , Giant Cells/drug effects , Glycerol/pharmacology , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycoproteins/pharmacology , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Humans , Hydrogen-Ion Concentration , Interferon Inducers/chemistry , Lectins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TemperatureABSTRACT
During a chemotaxonomic survey of Calophyllum extracts present in the National Cancer Institute's natural product repository, four new pyranocoumarins were isolated from extracts of C. lanigerum var. austrocoriaceum and C. teysmannii var. inophylloide (King.) P. F. Stevens (Clusiaceae). The structure elucidation and anti-HIV activity of calanolide E2 (4), cordatolide E (5), pseudocordatolide C (6), and calanolide F (9), along with a simple prenylated coumarin precursor (11), are described here.
