Subject(s)
Attitude of Health Personnel , Nursing Staff, Hospital/psychology , Perioperative Nursing , Adult , Data Collection , Female , Humans , Japan , Male , Middle Aged , WorkABSTRACT
Various lectins have attracted attention as potential microbicides to prevent HIV transmission. Their capacity to bind glycoproteins has been suggested as a means to block HIV binding and entry into susceptible cells. The previously undescribed lectin actinohivin (AH), isolated by us from an actinomycete, exhibits potent in vitro anti-HIV activity by binding to high-mannose (Man) type glycans (HMTGs) of gp120, an envelope glycoprotein of HIV. AH contains 114 aa and consists of three segments, all of which need to show high affinity to gp120 for the anti-HIV characteristic. To generate the needed mechanistic understanding of AH binding to HIV in anticipation of seeking approval for human testing as a microbicide, we have used multiple molecular tools to characterize it. AH showed a weak affinity to Man alpha(1-2)Man, Man alpha(1-2)Man alpha(1-2)Man, of HMTG (Man8 or Man9) or RNase B (which has a single HMTG), but exhibited a strong and highly specific affinity (K(d) = 3.4 x 10(-8) M) to gp120 of HIV, which contains multiple Man8 and/or Man9 units. We have compared AH to an alternative lectin, cyanovirin-N, which did not display similar levels of discrimination between high- and low-density HMTGs. X-ray crystal analysis of AH revealed a 3D structure containing three sugar-binding pockets. Thus, the strong specific affinity of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the "cluster effect" of lectin. Thus, AH is a good candidate for investigation as a safe microbicide to help prevent HIV transmission.
Subject(s)
Bacterial Proteins/pharmacology , HIV Fusion Inhibitors/pharmacology , HIV Infections/prevention & control , HIV-1/drug effects , Lectins/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacokinetics , Binding Sites , Carrier Proteins/pharmacokinetics , Carrier Proteins/pharmacology , Crystallography, X-Ray , HIV Envelope Protein gp120/chemistry , HIV Fusion Inhibitors/chemistry , HIV Fusion Inhibitors/pharmacokinetics , HIV-1/pathogenicity , HIV-1/physiology , Humans , In Vitro Techniques , Kinetics , Lectins/chemistry , Lectins/pharmacokinetics , Mannose/chemistry , Mannosides/chemistry , Models, Molecular , Protein Structure, TertiaryABSTRACT
Mycophenolic acid (MPA) was identified as an inhibitor of syncytium formation during the screening of human immunodeficiency virus (HIV) entry inhibitors. MPA is a well-known inhibitor of inosine monophosphate dehydrogenase and anti-HIV activity has been reported in vitro and in vivo. MPA inhibited syncytium formation in T cell-tropic and macrophage-tropic systems with IC50 values of 0.1 and 0.5 microM, respectively. The reduction of HIV gp120 expression by MPA (1.0 microM) was observed by use of Western blot analysis. Furthermore, the addition of guanosine restored both syncytium formation and gp120 expression in the presence of MPA. These results suggest that MPA inhibits not only reverse transcription by depletion of a substrate, GTP, as has been reported, but also syncytium formation through a predominant reduction in the amount of gp120 that is vigorously expressed in the above transformed cells and may be in HIV-infected cells.
Subject(s)
Antiviral Agents/pharmacology , Gene Expression/drug effects , Giant Cells/drug effects , HIV Envelope Protein gp120/metabolism , HIV-1/drug effects , Mycophenolic Acid/pharmacology , Blotting, Western , Enzyme Inhibitors/pharmacology , Genes, Reporter/genetics , Giant Cells/physiology , HIV-1/metabolism , HeLa Cells , Humans , Mycophenolic Acid/chemistryABSTRACT
A phenalenone compound, atrovenetinone methyl acetal, was isolated from a culture broth of Penicillium sp. FKI-1463 as an HIV-1 integrase inhibitor, and it showed anti-HIV activity in vitro. HIV-1 integrase inhibition and anti-HIV activity of two other natural phenalenones were also studied. Among the tested compounds, funalenone inhibited HIV-1 integrase with an IC50 value of 10 microM and showed the best selectivity (anti-HIV, IC50=1.7 microM; cytotoxicity, IC50=87 microM).
Subject(s)
Antifungal Agents/pharmacology , Enzyme Inhibitors/pharmacology , HIV Integrase/drug effects , Ketones/pharmacology , Naphthalenes/pharmacology , Phenalenes/chemistry , Polycyclic Compounds/pharmacology , Antifungal Agents/chemistry , Enzyme Inhibitors/chemistry , Ketones/chemistry , Naphthalenes/chemistry , Phenalenes/pharmacology , Polycyclic Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Actinohivin (AH) is a potent anti-human immunodeficiency virus (HIV) protein that consists of highly conserved three-tandem repeats (segments 1, 2, and 3). The molecular target of AH in its anti-HIV activity is high-mannose-type saccharide chains of HIV gp120. This article deals with sequence requirements for the anti-HIV activity of AH. The deleted or substituted DNAs encoding AH or His-AH were prepared using mutagenic oligonucleotide primers in PCR. The mutant constructs were expressed in Escherichia coli, and the activities of the recombinant protein products were examined by a syncytium-formation assay system that mimics anti-HIV activity. The single segment mutant His-AHs showed no anti-syncytium-formation activity, but the mutant His-AHs, which consists of 2 or 3 segments, retained reduced activities. His-AH(6-114) dramatically reduced the anti-syncytium-formation activity to that of His-AH(36-114) or His-AH(I5A). Furthermore, His-AH(Q33A), His-AH(Q71A), and His-AH(Q109A) in which glutamine residues were substituted into alanine showed reduced activities of 1/20, 1/10, and 1/30, respectively, in anti-syncytium formation compared with His-AH. These results indicate that three segments of AH are necessary for potent anti-syncytium-formation activity-that is, for potent anti-HIV activity and the cooperated involvement of each segment of AH increased the AH-gp120 interaction.
Subject(s)
Actinobacteria/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/pharmacology , Carbohydrate Metabolism , HIV/drug effects , Amino Acid Sequence , Amino Acid Substitution/genetics , Anti-HIV Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Conserved Sequence , Cysteine/genetics , Cysteine/metabolism , Enzyme-Linked Immunosorbent Assay , Giant Cells/drug effects , HIV/physiology , HeLa Cells , Humans , Inhibitory Concentration 50 , Molecular Sequence Data , Sequence Deletion/geneticsABSTRACT
We searched human immunodeficiency virus (HIV) entry inhibitors and found a novel anti-HIV protein, actinohivin (AH), in a culture filtrate of the newly discovered genus actinomycete Longispora albida gen. nov., sp. nov. This paper deals with the mechanism of action of the anti-HIV activity of AH. AH exhibited potent anti-HIV activities against various strains of HIV-1 and HIV-2. AH bound to the glycoprotein gp120 of various strains of HIV-1 and gp130 of simian immunodeficiency virus (SIV), but did not bind to non-glycosylated gp120 nor to cells having CD4 and coreceptors, suggesting that AH inhibits viral entry to cells by binding to the envelope glycoprotein. The investigation of the effects of various sugars on AH-gp120 binding by ELISA revealed that yeast mannan alone strongly inhibited the binding (IC50 = 3.0 microg/ml). Experiments investigating the binding of AH to other glycoproteins revealed that AH binds to ribonuclease B and thyroglobulin that have a high-mannose type saccharide chain, but not to other glycoproteins having a N-glycoside type saccharide chain. The above results indicate that high-mannose type saccharide chains of gp120 are molecular targets of AH in its anti-HIV activity.
