Human adenoviruses are of major medical interest because they cause respiratory infections in children and young adults and because they are bonafide tumor viruses. Fiber is a glycoprotein important to a number of adenovirus functions, including assembly of virus particles, attachment of virus to the cell surface, and antigenicity. Fiber is synthesized in the cytoplasm and then transported to the necleus for incorporation into virions; the details of its glycosylation and transport are not well characterized. Fiber is composed of three domains: an N- terminal tail, an internal shaft, and a C-terminal knob. The N- terminal tail apparently is responsible for binding to penton base in the assembly of virions; the C-terminal knob is thought to be responsible for binding to a specific cell receptor and contains an adenovirus type-sepcific antigen. When the complete DNA sequence of fiber genes from Ad2, Ad3, and Ad7 are compared, two short regions of complete homology in predicted amino acide sequence can be identified. This proposal will investigate the glycosylation and transport of the fiber protein to the nucleus. The hypothesis to be tested is that these conserved sequences play an important role in one or more aspects of fiber biochemistry: by specifying signals for transport to the nucleus, by binding to penton base during virus assembly, by serving as sites for glycosylation, or by recognizing the cell fiber, particularly within the conserved amino acid sequences, will be tested for their effects on these processes. These mutants will be characterized for transport to the nucleus by indirect immunofluorescence. In order to express mutated fiber proteins, transient expression systems will be tested in CVI, COS, and HeLa cells. Competitive binding experiments using either deletions of fiber or specific synthetic polypeptides to attempt to block fiber binding to penton base or to the cell receptor will be used to map the important regions for these processes. The sites of glycosylated of Ad2 and Ad7 fiber will be mapped by determining the positions of glycosylated proteolytic fragments within the fiber molecule. These early studies will lead to further investigations of the steps in the glycosylation and transport of pathways for fiber, as a model for glycoproteins targeted to the nucleus.
NIH ExPorter
NIH ExPorter