Research into antivirals continues to be a major focus worldwide. As resistance to current drugs increases, new medications are needed. In addition, new viruses and strains of viruses continue to spring up and evolve which often necessitate the need for new treatments. The research outlined here focuses on a cyanobacterial lectin which has shown potency against HIV and has the potential to neutralize other viruses as well. The compound, Cyanovirin-N (CV-N), is a lectin which acts like a pseudo antibody and has shown antiviral activity against enveloped viruses including HIV, influenza hemagglutinin (HA) and Ebola glycoprotein. The researchers involved in this study were interested in determining how this lectin, using variants of different stability that they designed, binds, and how certain functional groups affect binding. They used several techniques, including Surface Plasmon Resonance (SPR), to uncover this information.1

Many viruses have a viral envelope or outer layer which can provide protection to the virus when invading a host. The viral envelope is often composed of a portion of the host cell membrane (proteins and phospholipids) as well as viral glycoproteins, which allow the virus to evade detection by the host immune system. Enveloped viruses include, but are not limited to, hepatitis and coronavirus.2

Cyanovirin-N (CV-N), the protein, studied here, is a natural product as it is produced by cyanobacteria (also known as blue-green algae). It has demonstrated inhibitory properties against several enveloped viruses including human immunodeficiency virus and the Ebola virus. CV-N is not cytotoxic, so it is considered a potential glycan-targeting agent for prevention of seasonal and pandemic influenza diseases.1 Lectins like CV-N bind to carbohydrates via exposed sugar groups. The multivalent binding character of cyanobacterial lectins allows for direct binding to trimeric envelope glycoproteins at low surface density and reversible binding with glycoproteins linked to the cell membrane.1 Researchers studied CV-N variants in terms of both kinetics and thermodynamics for interactions with influenza related targets.

Experimental

BACKGROUND
Researchers were interested in better understanding the antiviral CV-N which is known to combat the replication of envelope proteins like HIV gp120, influenza hemagglutinin and Ebola glycoprotein.1 They varied chemistry like the mannose-mannose linkages in the target and used CV-N derivatives and oligomers as the analyte to determine their effects on binding. 

Conditions

 
 Instrument:
SR7500DC (2-channel SPR)
 Sensor Chip:  Dextran (CMD500D; Xantec)
 Temperature:  25 C
 Target:  Recombinant influenza A virus hemagglutinin H3 protein (HA), Dimannosylated peptide P2 (DM) and monomannosylated peptide P3 (MM)
 Analyte:  soluble dimeric head-to-tail linked tandem CV-N (CVN2L0) (4 disulfide bonds), and variants V2 (3 disulfide bonds), and V4 and V5 (2 disulfide bonds each)
Running Buffer 10µM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween, pH 7.4
 Association Time: 2.5 minutes
Dissociation Time 5 minutes Flow Rate: 30 μL/min
 Regeneration: 10 mM glycine pH 1.5

Results

Researchers studied dimeric head-to-tail linked tandem CV-N (CVN2L0) which has a native high-affinity binding site for oligosaccharides with high-mannose content and previously showed enhanced HIV neutralization compared to wild-type CV-N. The structure is such that it contains two intramolecular disulfide bonds forming a monomer, or an intertwined dimer. They recombinantly expressed CVN2L0 and variants with less than four Cys– Cys bridges and studied recognition through the known high affinity Mana(1-->2)Mana unit using chemically prepared (di-) mannosylated peptides derived from the HA head domain for binding CV-N and disulfide bridge variants. They also symmetrically replaced native Cys residues using previously found multi-specific mutations. The short peptides were chemically linked with mannose or dimannose to determine binding affinities to CVN2L0 and several variants. As the number of disulfide bridges near the glycan pocket decreased, the affinity decreased (weaker binding). They also varied the number of mannose-mannose linkages in the target. Affinity increased (tighter binding) as the number of mannose- mannose linkages increased and there was essentially no binding to the mono-mannosylated peptide with a N- terminal cysteine.1

An example of binding results obtained for a V2 variant of soluble CV-N with 3 disulfide bonds to immobilized HA is shown here:



Figure 1: Concentrations of the CV-N ranged from 31.2 nM to 1 ㎛. The data is fit to a conformational change model in ClampTM with a KD1 of 49 nM and a KD2 of 8 ㎛.

 

Summary

  • Both wild type CV-N and its variants bind to glycoproteins found on enveloped viruses. And, binding affinity changes when the number of disulfide bridges near the glycan pocket is varied and by changing the number of mannose-mannose linkages on the 1
  • Mannosylated peptides developed in this study can be used as protein scaffolds for screening binding characteristics of antiviral agents by 1
  • Based on computational protein design, researchers made new glycan-interacting homodimeric CVN2L0 scaffolds that can be used to further probe binding capacities at CV-N low affinity binding 1
  • Because lectins and pseudo antibodies bind highly conserved epitopes on the influenza HA protein, they could play a key role in the rational design of next generation vaccines for both preventative and therapeutic 1