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The broadly neutralizing antibody 2G12 recognizes a conserved cluster of high-mannose glycans on the surface envelope spike of HIV, suggesting that the "glycan shield" defense of the virus can be breached and may, under the right circumstances, serve as a vaccine target. In an attempt to recreate features of the glycan shield semisynthetically, oligomannosides were coupled to surface lysines on the icosahedral capsids of bacteriophage Q beta and cowpea mosaic virus (CPMV). The Q beta glycoconjugates, but not CPMV, presented oligomannose clusters that bind the antibody 2G12 with high affinity. However, antibodies against these 2G12 epitopes were not detected in immunized rabbits. Rather, alternative oligomannose epitopes on the conjugates were immunodominant and elicited high titers of anti-mannose antibodies that do not crossreact with the HIV envelope. The results presented reveal important design considerations for a carbohydrate-based vaccine component for HIV., ((c) 2010 Elsevier Ltd. All rights reserved.)

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.

Cowpea mosaic virus is composed of 60 identical copies of a two-subunit protein organized in pentameric assemblies around the icosahedral 5-fold symmetry axis. Treatment of the virus with the Ni(II) complex of the tripeptide GGH and a peroxide oxidant, or irradiation in the presence of Ru(bpy)(3)(2+) and persulfate generates covalent crosslinks across the pentameric subunit boundaries, effectively stitching the subunits together. Intersubunit crosslinking was found to occur exclusively at adjacent tyrosine residues (Y52-Y103), as predicted from the X-ray crystal structure of the capsid, and to be more extensive with the photochemical ruthenium system. The Ni/GGH oxidative procedure was also used to make covalent attachments to the virion by trapping with a functionalized disulfide reagent.

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.

Wild-type Cowpea mosaic virus (CPMV) displays no cysteine side chains on the exterior capsid surface and is therefore relatively unreactive with thiol-selective reagents. Four CPMV mutants bearing cysteine residues in one of two exterior positions of the asymmetric unit were created. The mutants were shown to aggregate by virtue of disulfide bond formation in the absence of added reducing agent, bind to metallic gold, and undergo selective reactions at the introduced thiol residues. Controlled aggregation by virtue of biotin-avidin interactions was demonstrated, as was the independent derivatization of reactive lysine and cysteine positions. The ability to introduce such reactivity into a system that can be readily prepared and isolated in gram quantities should open new doors to applications in biochemistry, materials science, and catalysis.