Characterization and nuclear localization of the fiber protein encoded by the late region 7 of bovine adenovirus type 3

Characterization and nuclear localization of the fiber protein encoded by the late region 7 of... To identify the protein encoded by the L7 region of bovine adenovirus-3 (BAdV-3), specific antisera were raised by immunizing rabbits with bacterial fusion proteins encoding the N-terminus or C-terminus of the BAdV-3 fiber protein. Immunoprecipitation and Western blot analysis confirmed that the fiber is expressed as a 102 kDa glycoprotein, which is localized to the nucleus of infected cells. To identify the nuclear localization signals (NLS), BAdV-3 fiber deletion mutants and GFP/β-galactosidase fusion proteins were expressed in transfected cells, and subcellular localization was visualized by immunofluorescence microscopy. Analysis of deletion mutants localized the NLS to the N-terminal 41 amino acids. Analysis of the N-terminal 41 amino acids identified a cluster of basic residues between amino acid 14 and 20. Substitution of the basic residues ( 16 KAKR 19 ) with acidic residues ( 16 EAEE 19 ) resulted in the accumulation of fiber in the cytoplasm. However, 16 KAKR 19 or 12 VYPYKAKRPNI 22 were not sufficient for efficient transport of a cytoplasmic protein GFP/β-galactosidase to the nucleus. The recombinant BAdV-3 expressing mutant fiber containing 16 EAEE 19 instead of 16 KAKR 19 was unable to replicate efficiently in Madin-Darby bovine kidney cells, suggesting that the NLS of fiber carries out important in vivo functions. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Characterization and nuclear localization of the fiber protein encoded by the late region 7 of bovine adenovirus type 3

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Publisher
Springer Journals
Copyright
Copyright © 2004 by Springer-Verlag/Wien
Subject
LifeSciences
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-004-0323-x
Publisher site
See Article on Publisher Site

Abstract

To identify the protein encoded by the L7 region of bovine adenovirus-3 (BAdV-3), specific antisera were raised by immunizing rabbits with bacterial fusion proteins encoding the N-terminus or C-terminus of the BAdV-3 fiber protein. Immunoprecipitation and Western blot analysis confirmed that the fiber is expressed as a 102 kDa glycoprotein, which is localized to the nucleus of infected cells. To identify the nuclear localization signals (NLS), BAdV-3 fiber deletion mutants and GFP/β-galactosidase fusion proteins were expressed in transfected cells, and subcellular localization was visualized by immunofluorescence microscopy. Analysis of deletion mutants localized the NLS to the N-terminal 41 amino acids. Analysis of the N-terminal 41 amino acids identified a cluster of basic residues between amino acid 14 and 20. Substitution of the basic residues ( 16 KAKR 19 ) with acidic residues ( 16 EAEE 19 ) resulted in the accumulation of fiber in the cytoplasm. However, 16 KAKR 19 or 12 VYPYKAKRPNI 22 were not sufficient for efficient transport of a cytoplasmic protein GFP/β-galactosidase to the nucleus. The recombinant BAdV-3 expressing mutant fiber containing 16 EAEE 19 instead of 16 KAKR 19 was unable to replicate efficiently in Madin-Darby bovine kidney cells, suggesting that the NLS of fiber carries out important in vivo functions.

Journal

Archives of VirologySpringer Journals

Published: Sep 1, 2004

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