Microbial Antiviral Proteins with GBP Activity
Antiviral compounds made by eukaryotes that recognize unique glycan determinants represent a new paradigm in terms of understanding the innate immune system of primitive organisms and how that might relate to mammalian innate immune systems. Many mammalian GBPs act as innate immune defenders, including the C-type lectins Ficolins/Mannose-binding protein and other collectins. Interestingly, many of these bind glycan determinants often relatively rich in mannose and/or fucose. One promising anti-HIV-1 drug in development is cyanovirin-N, initially isolated from an extract of the cyanobacterium Nostoc ellipsosoprum. While CVN was originally thought to be an orphan lectin with little homology to any other known protein family, a family of CVN homologs, termed CVNHs, has been described. Members of this family are found in multicellular ascomycetous fungi and in ferns and share a 3-D fold. A CVNH of the toxin-producing cyanobacterium Microcystis aeruginosa also binds high mannose-type glycans and is involved in cell–cell attachment of Microcystis.
Defining the glycan binding specificity and mode of action for virucidal lectins may help to develop new therapeutic approaches directed at combating viral infections.
Cyanovirin-N (Nostoc ellipsosporum - a cyanobacterium)
Cyanovirin-N (CVN) was chosen as a paradigm because of its relevance to human disease and as an example of a new virucidal found in nature. CVN was originally isolated from the cyanobacterium Nostoc ellipsosporum, in a screening program for anti-HIV activities. CVN is a small protein of 101 amino acids with two internal tandem repeats of ~50 amino acid. Its structure established a novel fold with no significant similarity to any other known protein. It exhibits pseudo-symmetry and comprises two domains, each possessing an independent glycan binding site. The protein can also exist as a domain-swapped dimer. CVN inhibits HIV entry into cells by interacting with the high mannose-type N-glycans on the envelope glycoprotein gp120 of HIV-1. CVN also binds to the glycoproteins of other enveloped viruses, such as SIV, Ebola, influenza and hepatitis C. Thus, CVN represents a new paradigm of microbial GBPs, wherein a unique glycan binding domain comprised of approximately 50 amino acids, exhibits specificity toward α1-2-linked mannose residues.
CFG Participating Investigators contributing to the understanding of this paradigm
CFG Participating Investigators (PIs) who have contributed to studies of this paradigmatic protein include: Simone Ottonello (University of Parma, Italy) and Angela M. Gronenborn (University of Pittsburgh, USA).
Progress toward understanding this GBP paradigm
The physiological ligand for Cyanovirin-N is not precisely known. However, based on extensive data on carbohydrate binding studies on this protein by NMR, X-ray, and glycan microarray screening on the CFG microarray, it is expected that CV-N would recognize any glycans that contain highly enriched alpha (1->2) linkage - mannoses.
Cellular expression of GBP and ligands
The Cyanovirin-N protein is expressed by Cyanobacterium (blue-green alga) Nostoc ellipsosporum. It binds ligands on glycoproteins of enveloped viruses including HIV, SIV, Ebola, influenza and hepatitis C.
Biosynthesis of ligands
The envelope glycans of immunodeficiency virions, that are ligands for CV-N and the CVNH family members, are almost entirely oligomannose glycans. There are radical differences between recombinant and viral gp120 envelope glycosylation. The glycosylation of recombinant gp120 reveals an unprocessed intrinsic patch of Man5–9GlcNAc2 glycans, which appears to have resisted α-mannosidase activity. The remaining glycans in the recombinant gp120 are a cell-speciﬁc mixture of complex type glycans. The trimeric viral gp120 contains the original mannose patch. The array of complex glycans is absent in the native viral trimeric gp120 that contains mostly high mannose (Man5–9GlcNAc2) glycans.
Below are a few representations for the available structures of CV-N determined so far:
The solution structure of wild type CV-N as a monomer (PDB:2EZM).
The crystal structure of P51G mutant of CV-N as a swapped dimer (PDB:1L5B).
The crystal structure of swapped-dimeric CV-N in complex with hexamannose (PDB:3GXY).
Biological roles of GBP-ligand interaction
In vitro, low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus.
CFG resources used in investigations
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for "cyanovirin-N".
Analysis has not been conducted. CVN is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
Knockout mouse lines
The CFG has contributed glycans for various glycan specificity studies. Glycan specificity analysis has been conducted for Cyanovirin-N using the CFG glycan microarray as shown below. To see all glycan array results for Cyanovirin-N, click here.
A large family of CVNHs has been found in both eukaryotic fungi and cyanobacteria (see refs 3, 4 and 5 below). Click here for CFG data on CVNHs.
- ↑ 1.0 1.1 1.2 Boyd, M.R., Gustafson, K.R., McMahon, J.B., Shoemaker, R.H., O'Keefe, B.R., Mori, T., Gulakowski, R.J., Wu, L., Rivera, M.I., Laurencot, C.M., Currens, M.J., Cardellina, J.H., 2nd, Buckheit, R.W., Jr., Nara, P.L., Pannell, L.K., Sowder, R.C., 2nd and Henderson, L.E. 1997. Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development. Antimicrob Agents Chemother, 41, 1521-1530
- ↑ 2.0 2.1 Bewley, C.A., Gustafson, K.R., Boyd, M.R., Covell, D.G., Bax, A., Clore, G.M., and Gronenborn, A.M. 1998. Solution structure of cyanovirin-N, a potent HIV-inactivating protein. Nat Struct Biol 5, 571-578
- ↑ Percudani, R., Montanini, B. and Ottonello, S. 2005. The anti-HIV cyanovirin-N domain is evolutionarily conserved and occurs as a protein module in eukaryotes. Proteins, 60, 670-678
- ↑ Koharudin, L.M., Viscomi, A.R., Jee, J.G., Ottonello, S. and Gronenborn, A.M. 2008. The evolutionarily conserved family of cyanovirin-N homologs: structures and carbohydrate specificity. Structure, 16, 570-584
- ↑ Kehr, J.C., Zilliges, Y., Springer, A., Disney, M.D., Ratner, D.D., Bouchier, C., Seeberger, P.H., de Marsac, N.T. and Dittmann, E. 2006. A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa. Mol Microbiol, 59, 893-906
- ↑ Bewley, C.A., Kiyonaka, S., and Hamachi, I. (2002). Site-specific discrimination by cyanovirin-N for alpha-linked trisaccharides comprising the three arms of Man(8) and Man(9). J Mol Biol 322, 881-889
- ↑ Barrientos, L.G., Matei, E., Lasala, F., Delgado, R., and Gronenborn, A.M. (2006). Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition. Protein Eng Des Sel 19, 525-535
- ↑ Yang, F., Bewley, C.A., Louis, J.M., Gustafson, K.R., Boyd, M.R., Gronenborn, A.M., Clore, G.M., and Wlodawer, A. (1999). Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. J. Mol. Biol. 288, 403-412
- ↑ Barrientos, L.G., Louis, J.M., Botos, I., Mori, T., Han, Z., O'Keefe, B.R., Boyd, M.R., Wlodawer, A., and Gronenborn, A.M. (2002). The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures. Structure 10, 673-686
- ↑ Doores, K.J., Bonomelli, C., Harvey, D.J., Vasiljevic, S., Dweck, R.A., Burton, D.R., Crispin, M., and Scanlan, C.N. (2010) Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc. Natl. Acad. Sci. U.S.A. 107: 13800-13805
The CFG is grateful to the following PIs for their contributions to this wiki page: Angela Gronenborn