Cyanovirin-N A sugar-binding antiviral protein Christian García Blanca 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 &

Cyanovirin Structure Domains: A = residues 1-38 & Domain A

Cyanovirin Structure Domains: A = residues 1-38 & B = residues Domain B

Cyanovirin Structure Domains: A = residues 1-38 & B = residues Each domain has triple stranded β sheet with β hairpin linked by a helix.

Cyanovirin Structure Domains: A = residues 1-38 & B = residues 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 ( Man  1-2Man  ) 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-9Hexamannose

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.