Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability.

Irene Maier,Georg Kontaxis,Robert H Schiestl

doi:10.3390/molecules26123621

Abstract

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV–N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV–N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV–N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV–N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.

Highlights

The ability of lectins, such as cyanovirin-N (CV–N), and antiviral carbohydratebinding agents to bind highly conserved epitopes on the influenza hemagglutinin (HA)glycoprotein is of key importance to the rational design of vaccines for prophylactic and therapeutic use [1,2,3]
The low binding affinities of anti-human immunodeficiency virus (HIV) neutralizing Ab (NAb) 2G12 to the influenza HA full-length protein and Ebola GP1,2 were evaluated in the μ-molar range for avidity as compared to the binding of HIV-1 gp120, and CV–N binding to viral GPs
We examined the binding affinities and avidity of anti-HIV neutralizing 2G12 to an influenza HA full-length protein and Ebola GP1,2 in the mid μM range (Figure 1)

Summary

Introduction

The ability of lectins, such as cyanovirin-N (CV–N), and antiviral carbohydratebinding agents to bind highly conserved epitopes on the influenza hemagglutinin (HA)glycoprotein is of key importance to the rational design of vaccines for prophylactic and therapeutic use [1,2,3]. N-linked glycosylation is necessary and sufficient for tetherin-mediated restriction of HIV-1 release [7], but it remains controversial in several other studies. The importance of using defined glycosylation patterns has been demonstrated in vitro, which in turn depends on the conformation of the surface-expressed HA membrane-fusion active part in the latter [8]. The surface-expressed envelope protein HA and the neuraminidase on influenza A virus are known for tetherin antagonism in a strain-specific manner [9], as they facilitate recognition of host receptor binding sites [10,11]. To study the glycosylation pattern, a binding-site specific tool such as the domain-swapped anti-HIV 2G12 antibody (Ab) attachment had to be used. The domain-swapped anti-HIV neutralizing Ab (NAb) complexed with Man(9)GlcNAc(2), was co-crystallized with Man(4), Man(5), Man(7), and

Methods

Results

Conclusion