Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX.
Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides.
Zakhartchouk
Alexander
A
Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E3.
Connors
Wayne
W
van Kessel
Andrew
A
Tikoo
Suresh Kumar
SK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D017921:Mastadenovirus / Q000235:genetics* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D036022:Capsid Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C018315:hexon capsid protein, Adenovirus; C054562:yellow fluorescent protein, Bacteria