Deletion analysis of functional domains in baculovirus-expressed adenovirus type 2 fiber.
Various forms of Ad2 fiber were expressed in insect cells using recombinant baculoviruses and phenotypically characterized with respect to the following properties: trimerization, binding to penton base, nuclear targeting, and glycosylation. The morphology and dimensions of full-length fiber produced by invertebrate cells were indistinguishable from those observed in extracts from lytically infected mammalian cells. The domain required for trimer formation was mapped to the C-terminus, between amino acids 541 and 582. The N-terminal domain, between amino acids 1 and 16, negatively influenced the trimerization efficiency. Fiber gene products reduced to the shaft portion of the fiber capsomer formed significant amounts of stable dimers. Recognition with penton base only occurred with trimeric forms of fiber and was apparently not affected by deletion of the first 60 amino acids from the N-terminus. Fiber deleted of the Met1-Gly60 sequence was found to localize within the nucleus at levels similar to those of full-length fiber. All recombinant fibers, including tail-and-know-deleted forms, were found to be glycosylated using three separate assays, (i) in vivo labeling with [3H]glucosamine, (ii) binding to WGA, and (iii) reaction with monoclonal antibody RL2 directed against O-GlcNAc-containing glycopeptide. This implied that Ad2 fiber is a substrate for GlcNAc O-seryl transferase in insect cell cytoplasm and that at least one major glycosylation site is located in the shaft domain, between Met61 and Asn410.
Laboratoire de Virologie et Pathogénèse Moléculaires, Faculté de Médecine, Montpellier, France.
A
A
Novelli
P A
PA
Boulanger
D000260:Adenoviruses, Human / Q000235:genetics* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005814:Genes, Viral; D006031:Glycosylation; D007313:Insecta; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016364:Reading Frames; D011995:Recombination, Genetic; D014162:Transfection
D036022:Capsid Proteins; D009838:Oligodeoxyribonucleotides; C018315:hexon capsid protein, Adenovirus