1348 ¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/cbic.200300759 ChemBioChem 2003, 4, 1348±1351 Icosahedral Virus Particles as Polyvalent Carbohydrate Display Platforms Krishnaswami S. Raja, Qian Wang, and M. G. Finn* [a] Icosahedral viruses can serve as robust and programmable scaffolds to which a wide variety of chemical and biological structures may be attached. [1] Their advantages include bio- compatibility, the display of functional groups on both the inside and outside surfaces of the viral coat protein ; very large size (on the chemical scale) allowing for the independent attachment of different units to noninteracting positions and their presentation to the environment on dimensions relevant to those of biological cells; and the ability to introduce amino acid residues with unique reactivity in designed locations of the capsid structure. No other type of scaffold of comparable size is avail- able for which the structure is known to near atomic resolution. Among other applica- tions, we seek to employ virus particles to display biochemical units of importance to cellular recognition and signaling events. [2] Carbohydrates are an obvious choice in this regard, as their interactions with receptors mediate a variety of important processes. [3] A central theme in this area is polyvalen- cy. [4] The display of multiple copies of carbohydrates on polymeric, [5] dendritic, [6] small-molecule, [7] and other [8] supports has been shown to provide tighter binding to cell-surface receptors and/or more effective biochemical response than is observed with monomeric carbohydrate derivatives in sol- ution. [4] We describe here the covalent decoration of cowpea mosaic virus (CPMV) with sugar molecules, and studies of the binding behavior of the resulting multivalent particles with both carbohydrate-binding protein and cells. We have previously established that each asymmetric unit of wild-type CPMV contains one lysine residue that exhibits diminished conjugate acid pK a , identified as K38 of the small subunit. This residue is found on the exterior surface of the capsid and can be selectively targeted by isothiocyanate reagents, [11] such that 90 % or more of isothiocyanate attach- ments are made at this position, up to a value of 60 per virion (the total number of K38 residues). Thus, a-D-mannopyranosyl- phenyl-isothiocyanate (1) was reacted with wild-type CPMV at a molar ratio 500:1 (1 with respect to the concentration of viral protein) in 0.1 M potassium phosphate buffer (pH 7.0) containing 20 % DMSO (Scheme 1). Previous experiments with fluorescein isothiocyanate (FITC) established an average covalent loading under these conditions of 50  5 dye molecules per virus particle. The level of mannose loading on the product (2b) was measured with a modified fluorescamine assay [12] as 42  5 per virion, comparable to the result with the dye reagent. In all cases, derivatized CPMV samples were purified by dialysis and size-exclusion chromatography, and isolated in  60 % yield. In order to obtain a more heavily mannose-labeled particle, the above reaction was performed in sodium bicarbonate buffer (pH 9.0) containing 20 % DMSO (Scheme 1). Product 2a was found by fluorescamine assay to bear 120  12 mannose units per virion; again this is consistent with previous FITC observa- tions. CPMV derivatives 3a ± c, bearing both carbohydrate and dye molecules, were similarly constructed by using a virus mutant with one cysteine residue per asymmetric unit on the exterior surface, in addition to the normal reactive lysines (Scheme 1). [9e, 11b] Fluorescein bromoacetamide was first used to address the highly accessible cysteine side chains, and the purified intermediate particles were then decorated with mannose as above. Virions bearing fewer than 60 mannose units (2b, 2c, 3b, 3c) therefore have those carbohydrates displayed at the K38 positions. Since there are 60 component proteins per particle, samples 2a and 3a have approximately half of their 120 carbohydrates attached to lysine side chains [a] Prof. M. G. Finn, Dr. K. S. Raja, Dr. Q. Wang Departments of Chemistry, The Skaggs Institute for Chemical Biology and the Center for Integrative Molecular Biosciences The Scripps Research Institute, 10550 North Torrey Pines Road La Jolla, CA 92037 (USA) Fax: ( 1)619-784-8850 E-mail: mgfinn@scripps.edu Scheme 1. 1) 20% DMSO in buffer, 4 8C, 48 h; 2) dialysis; 3) size-exclusion chromatography. Reaction a, yielding product 2a : pH 9.0, 500 equiv. 1; reaction b, yielding product 2b : pH 7.0, 500 equiv. 1; reaction c, yielding product 2c : pH7.0, 100 equiv. 1. 4) 20% DMSO in buffer, pH 7.0, 4 8C, 48 h; 5) Size-exclusion chromatography; 6) 20% DMSO, pH 9.0, 4 8C, 48 h; product a: 500 equiv. 1; product b: 200 equiv. 1; product c: 100 equiv. 1.