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

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

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

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  1. Cyanovirin-N A sugar-binding antiviral protein Christian García Blanca Arroyo

  2. 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.

  3. 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.

  4. Introduction: Cyanovirin & HIV Cyanovirin

  5. 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.

  6. Cyanovirin Structure • Domains: A = residues 1-38 & 90-101

  7. Cyanovirin Structure • Domains: A = residues 1-38 & 90-101 Domain A

  8. Cyanovirin Structure • Domains: A = residues 1-38 & 90-101 B = residues 39-89 Domain B

  9. 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.

  10. 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).

  11. 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).

  12. Cyanovirin Domain Swapping • Exists as a monomer or as a Domain Swapped Dimer. Monomer DS Dimer

  13. 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.

  14. Cyanovirin Sugar Binding • Two (primary & secondary) carbohydrate binding sites (monomer). • Bind primarily N-Linked High-mannose oligosaccharides.

  15. Cyanovirin SugarBinding • Primary site binding of dimannose (Mana1-2Mana) depends on the establishment of 8 hydrogen bonds.

  16. Cyanovirin Sugar Binding • Secondary site interface formed by 2 (hexamanose) or 3 (Man-9) a1-2 stacked rings. Man-9 Hexamannose

  17. 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.

  18. 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.

  19. 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.

  20. 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

  21. 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

  22. In vivo CV-N gel in macaque

  23. 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.

  24. 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.

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