title: Chemical conjugation of heterologous proteins on the surface of Cowpea mosaic virus.
abstract: Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.
authors.affiliation: Department of Molecular Biology, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Anju
authors.initials: A
authors.lastname: Chatterji
authors.forename: Wendy
authors.initials: W
authors.lastname: Ochoa
authors.forename: Lara
authors.initials: L
authors.lastname: Shamieh
authors.forename: Shant P
authors.initials: SP
authors.lastname: Salakian
authors.forename: Sek Man
authors.initials: SM
authors.lastname: Wong
authors.forename: Gail
authors.initials: G
authors.lastname: Clinton
authors.forename: Partho
authors.initials: P
authors.lastname: Ghosh
authors.forename: Tianwei
authors.initials: T
authors.lastname: Lin
authors.forename: John E
authors.initials: JE
authors.lastname: Johnson
mesh_terms: D001426:Bacterial Proteins; D017122:Bacteriophage T4 / Q000201:enzymology; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D002853:Chromatography, Liquid; D017798:Comovirus / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003432:Cross-Linking Reagents / Q000737:chemistry; D016503:Drug Delivery Systems; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009113:Muramidase / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D009113:Muramidase; D011506:Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; C095768:inlB protein, Listeria monocytogenes