Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin

Abstract

The novel antiviral protein cyanovirin-N (CV-N) was initially discovered based on its potent activity against the human immunodeficiency virus (HIV). Subsequent studies identified the HIV envelope glycoproteins gp120 and gp41 as molecular targets of CV-N. More recently, mechanistic studies have shown that certain high-mannose oligosaccharides (oligomannose-8 and oligomannose-9) found on the HIV envelope glycoproteins comprise the specific sites to which CV-N binds. Such selective, carbohydrate-dependent interactions may account, at least in part, for the unusual and unexpected spectrum of antiviral activity of CV-N described herein. We screened CV-N against a broad range of respiratory and enteric viruses, as well as flaviviruses and herpesviruses. CV-N was inactive against rhinoviruses, human parainfluenza virus, respiratory syncytial virus, and enteric viruses but was moderately active against some herpesvirus and hepatitis virus (bovine viral diarrhea virus) strains (50% effective concentration [EC(50)] = approximately 1 micro g/ml) while inactive against others. Remarkably, however, CV-N and related homologs showed highly potent antiviral activity against almost all strains of influenza A and B virus, including clinical isolates and a neuraminidase inhibitor-resistant strain (EC(50) = 0.004 to 0.04 micro g/ml). When influenza virus particles were pretreated with CV-N, viral titers were lowered significantly (>1,000-fold). Further studies identified influenza virus hemagglutinin as a target for CV-N, showed that antiviral activity and hemagglutinin binding were correlated, and indicated that CV-N's interactions with hemagglutinin involved oligosaccharides. These results further reveal new potential avenues for antiviral therapeutics and prophylaxis targeting specific oligosaccharide-comprised sites on certain enveloped viruses, including HIV, influenza virus, and possibly others.

FIG. 1.

Anti-influenza activity of CV-N in MDCK cells infected with influenza virus. Shown is a comparison of the concentration-dependent effects of CV-N on the viability of virus-infected cells (•) and control cells (○) infected with influenza virus A/Sydney/05/97 (H3N2) (A) and influenza virus B/Yamanashi/166/98 (B), as assessed by NR assay.

FIG. 2.

Binding of CV-N to influenza virus lysates. Shown is a comparison of the concentration dependent binding of Eu-CV-N to viral lysates of influenza A/PR/8/34 (H1N1) virus (•) and influenza A/Sydney/05/97 (H3N2) (○), as determined by a time-resolved fluorescence binding assay. Points are the averages ± standard deviations of six replicate determinations.

FIG. 3.

Analysis by immunoblotting of CV-N binding to influenza A virus lysates. Influenza A virus lysates from either strain A/PR/8/34 (H1N1) (CV-N resistant; PR8) or A/Sydney/05/97 (H3N2) (CV-N sensitive, Syd) were electrophoresed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were either stained with Coomassie blue (A) or blotted onto a PVDF membrane and incubated with either anti-HA antibodies (B) or CV-N-specific antibodies (C). Lane MW, molecular weight markers; lane HA, purified HA (A/Beijing/262/95 [H1N1]).

FIG. 4.

Binding of CV-N to purified HA isolated from influenza A/Beijing/262/95 (H1N1) virus. (A) Concentration-dependent binding of Eu-CV-N to purified HA as determined by time-resolved fluorescence. One hundred nanograms of CV-N is equivalent to 9.1 pmol of CV-N. (B) Concentration-dependent inhibition of CV-N binding to purified HA by the high-mannose oligosaccharide Man-8. Points are the averages ± standard deviations of four replicate determinations.