cyanovirin-n a sugar-binding antiviral protein christian garcía blanca arroyo
TRANSCRIPT
Cyanovirin-N
A sugar-binding antiviral protein
Christian GarcíaBlanca Arroyo
Introduction: Cyanovirin & HIV
• HIV infection of host cells is a stepwise process:
– Binding of Viral Envelope to CD4– Binding of Viral Envelope to other chemokines– Fusion of Viral and Host Cell membranes– Incorporation of viral DNA to host genome
• Cyanovirin is a compound capable of inhibiting the first two steps.
Introduction: Cyanovirin
• Cyanobacterial lectin with virucidal activity.
• Protein that irreversibly inhibits HIV entry.
• Molecular mechanism involves multivalent interactions with high-mannose oligosaccharides of viral envelope.
• NMR & X-ray Crystal structures resolved.
Introduction: Cyanovirin & HIV
Cyanovirin
Cyanovirin Structure
• Peptide 101 aa residues and less than 20% homology to any known protein.
• Two domain monomer is ~55Å by ~25Å
• Distant resemblance to the hyperthermophile DNA-binding protein Sac7d and to the SH3 domain of Spectrin.
Cyanovirin Structure
• Domains: A = residues 1-38 & 90-101
Cyanovirin Structure
• Domains: A = residues 1-38 & 90-101
Domain A
Cyanovirin Structure
• Domains: A = residues 1-38 & 90-101
B = residues 39-89
Domain B
Cyanovirin Structure
• Domains: A = residues 1-38 & 90-101
B = residues 39-89
• Each domain has triple stranded β sheet with β hairpin linked by a helix.
Cyanovirin Structure
• Domains: A = residues 1-38 & 90-101 B = residues 39-89
• Each domain has triple stranded β sheet with β hairpin linked by a helix.
• Two disulfide bonds (8:22 & 58:73).
Cyanovirin Structure
• Each domain has an oligosaccharide binding site.
• Stable structure & activity after: freezing, solvents, denaturants, detergents & boiling.
• High internal aa sequence homology (32% identical + 26% conservative).
Cyanovirin Domain Swapping
• Exists as a monomer or as a Domain Swapped Dimer.
Monomer
DS Dimer
Cyanovirin Domain Swapping
• DS Dimers exhibit identical structure to monomer except hinge region.
• DS Dimer is metastable and slowly converts to monomer (thermodynamically stable).
• Both Monomer & DS Dimer have equivalent antiviral activity.
Cyanovirin Sugar Binding
• Two (primary & secondary) carbohydrate binding sites (monomer).
• Bind primarily N-Linked High-mannose oligosaccharides.
Cyanovirin Sugar Binding
• Primary site binding of dimannose (Man1-2Man) depends on the establishment of 8 hydrogen bonds.
Cyanovirin Sugar Binding
• Secondary site interface formed by 2 (hexamanose) or 3 (Man-9) 1-2 stacked rings.
Man-9 Hexamannose
Cyanovirin Antiviral Activity
• Irreversible inactivation of diverse T-lymphocyte and Macrophage-tropic virus: (HIV-1, HIV-2, SIV, SHIV, FIV & Ebola).
• Inhibits fusion of HIV-infected & non-infected cells.
• Inhibits cell to cell transmission of HIV.
Cyanovirin Antiviral Activity
• Binding of GP120 is essential but not sufficient for virostatic activity.
• Other membrane proteins probably involved as CV-N interferes only with membrane bound GP120:CD4 complexes (but not soluble ones).
• CV-N can dissolve GP120:CD4 complexes.
• CV-N binding to GP120 surpasses the affinity of current mAb.
CV-N inhibits HIV infection
• in vitro and in vivo efficacy studies– human ectocervical– vaginal transmission models
• Evaluated a gel formulated CV-N as a topical vaginal microbicide against chimeric SHIV.
CV-N in vitro trials
• Explants were treated with CV-N before (-60 or -
5 min), simultaneously (0 min), or after (60 min) HIV challenge.
• Infections monitored by p24 ELISA & PCR
In vivo CV-N gel in macaque• 29 naive adult female m. fascicularis
• CV-N gel in cervicovaginal area.
• Pretreated medroxyprogesterone.
• For prophylactic study macaques received a single intravaginal dose.
• Five groups
– 6 macaques 0.5% CV-N– 6 macaques 1.0% CV-N– 6 macaques 2.0% CV-N– 4 macaques Placebo– 4 macaques Viral Controls
In vivo CV-N gel in macaque
Cyanovirin Trial
• Safety trial showed no adverse effects.
• Overall effectiveness of CV-N gels against vaginal SHIV infections around 85%.
• Macaques that did develop the infection likely subjected to vaginal trauma during procedure.
• Results suggest that CV-N is a promising agent as a topical vaginal microbicide for the prevention of sexual transmission of HIV infection.
Conclusions
• Physiological role of CV-N in cyanobacteria unknown.
• Promising for prevention & treatment of AIDS.
• CV-N mechanism overcomes proteomic approaches to vaccine development by targeting a relatively conserved GP.
• Prevents emergence of drug-resistant HIV strains by aborting the infectious process early on.