High-mannose-specific deglycosylation of HIV-1 gp120 induced by resistance to cyanovirin-N and the impact on antibody neutralization

Abstract

HIV-1 uses glycans on gp120 to occlude its highly immunogenic epitopes. To better elucidate escape mechanisms of HIV-1 from carbohydrate-binding agents (CBA) and to understand the impact of CBA-escape on viral immune evasion, we generated and examined the biological properties of HIV-1 resistant to cyanovirin-N (CV-N) or cross-resistant to additional CBAs. Genotypic and phenotypic characterization of resistant env clones indicated that 3-5 high-mannose residues from 289 to 448 in the C2-C4 region of gp120 were mutated and correlated with the resistance levels. The specificity and minimal requirements of deglycosylation for CV-N resistance were further assessed by mutagenesis study. The sensitivity of resistant variants to a range of CBAs, immunoglobulins, sera and monoclonal antibodies (MAb) were investigated. For the first time, our data have collectively defined the high-mannose residues on gp120 affecting CV-N activity, and demonstrated that CBA-escape HIV-1 has increased sensitivity to immunoglobulins and sera from HIV patients, and particularly to V3 loop-directed MAbs. Our study provides a proof-of-concept that targeting HIV-1 glycan shields may represent a novel antiviral strategy.

Cyanovirin-N potently inhibits human immunodeficiency virus type 1 infection in cellular and cervical explant models

“…In this way it was possible to determine kinetics and interaction half-life between the virus particles and the compound. CV-N was titrated on this platform for binding to HIV-1 BaL and HIV-1 RF , as well as a CV-Nresistant HIV-1 isolate (Hu et al, 2007). PHA was immobilized separately and used as a non-specific control lectin.…”

“…The variation in resonant frequency observed, a result of HIV-1 binding to the compound, allows determination of the kinetic parameters of the interaction. In these experiments HIV-1 BaL and HIV-1 RF were studied, along with a CV-N-resistant isolate of HIV-1 IIIB generated in vitro (Hu et al, 2007). HIV-1 RF demonstrated potent binding to CV-N (Fig.…”

“…CV-N, an 11 kDa protein originally purified from the cyanobacterium Nostoc ellipsosporum, potently inactivates a wide spectrum of HIV-1 strains and primary isolates, as well as a number of other viruses including HIV-2, simian immunodeficiency virus, Ebola and hepatitis C virus (Barrientos & Gronenborn, 2005;Boyd et al, 1997;Helle et al, 2006). The mechanism of action of CV-N involves multiple binding to high-mannose oligosaccharides predominantly in the C2-C4 region of the HIV-1 gp120 protein (Hu et al, 2007;Shenoy et al, 2001). This binding then inhibits the conformational change required for virus-target cell attachment and subsequent fusion.…”

“…Thus potent antiviral activity, lack of toxicity, resistance to physico-chemical denaturation as well as the unusual high genetic barrier to resistance (Balzarini et al, 2006;Hu et al, 2007;Witvrouw et al, 2005) suggest that CV-N is a promising anti-HIV molecule for use as a topical microbicide (Boyd et al, 1997;Esser et al, 1999).…”

The antiviral protein cyanovirin-N: the current state of its production and applications

“…The carbohydrate moiety of gp120 consists of 24 N-linked oligosaccharides, among which 11 are believed to be high-mannose or hybrid type, and 13 are complex type. CVN exerts its activity by binding to high-mannose oligosaccharides, located predominantly in the C2-C4 region of gp120, and prevents the virus entry by blocking fusion with the cell membrane and cell-to-cell transmission (Hu et al 2007;Shenoy et al 2001). The 1.35 A high-resolution structure of P51G-m4-CVN suggests that the Arg-76 and Glu-41 residues of CVN are critical components for the high mannose specificity and affinity.…”