Human adenoviruses are of major medical importance because they cause respiratory infections in children and young adults and because they are DNA tumor viruses. Fiber is a virus protein found at each vertex of the icosahedral capsid; it is important to a number of virus functions, including attachment to a cell surface receptor, assembly of infectious virions, and antigenicity. Fiber is thought to be a complex of three monomers, arranged into an N-terminal tail that binds to another capsid protein (penton base), an internal shaft, and a C-terminal knob that recognizes the cell surface receptor. Fiber from adenovirus serotypes 2 and 5 contains an unusual O-linked N-acetylglucosamine (O-GlcNAc), a saccharide modification found on many cytoplasmic and nuclear proteins (such as the proteins of the nuclear pore complex); O-GlcNAc may be important for a number of cellular functions, including assembly of multimeric complexes. These novel saccharides are added to fiber by a cytoplasmically oriented N-acetylglucosaminyltransferase shortly after synthesis of fiber molecules. Fibers from Ad2 and Ad5 also shared an epitope (recognized by antibody RL-2) with a nuclear pore protein (p62) and other O-GlcNAc-containing proteins. Fiber from Ad2 and Ad5 offers a unique system for the study of this O- GlcNAc modification and its potential roles in cellular processes, such as assembly of protein complexes. Ad2 and Ad5 fibers contain between 2 and 5 potential sites for O-GlcNAc attachment; amino acid sequencing of proteolytic fragments of fiber will be used to map the precise sites of attachment. A unique expression system, based on vaccinia virus recombinants, will be used to study mutants lacking one or more of the O- GlcNAc attachment sites. The proper assembly of fiber trimers can be monitored using a unique monoclonal antibody that recognizes only fiber trimers. Peptides containing the proposed sequences for O-GlcNAc attachment will be used to assay HeLa cell lysates for the N- acetylglucosaminyltransferase. Domains that encode the shared RL-2 epitope and that determine the ability to form trimers will be mapped. Domains in fiber that interact with penton base will also be mapped, using deletion and site-directed mutants made at the amino-terminus of fiber. A new form of cytoplasmic modification containing fucose and another carbohydrate have recently been detected by labeling of hexon and penton base. As a long term goal, this novel modification will be identified and its site of attachment mapped, using techniques similar to those described above for the study of O-GlcNAc. These studies may lead to the elucidation of the role of this newly detected modification on adenovirus nd other cellular proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038234-06
Application #
3294438
Study Section
Experimental Virology Study Section (EVR)
Project Start
1987-08-01
Project End
1995-03-31
Budget Start
1993-04-01
Budget End
1994-03-31
Support Year
6
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Hong, J S; Engler, J A (1996) Domains required for assembly of adenovirus type 2 fiber trimers. J Virol 70:7071-8
Michael, S I; Hong, J S; Curiel, D T et al. (1995) Addition of a short peptide ligand to the adenovirus fiber protein. Gene Ther 2:660-8
Hong, J S; Engler, J A (1991) The amino terminus of the adenovirus fiber protein encodes the nuclear localization signal. Virology 185:758-67
Mullis, K G; Haltiwanger, R S; Hart, G W et al. (1990) Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins. J Virol 64:5317-23
Hong, J S; Mullis, K G; Engler, J A (1988) Characterization of the early region 3 and fiber genes of Ad7. Virology 167:545-53