ADENOVIRUS 2 CODED EARLY GLYCOPROTEIN

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Proteins generally are localized at specific sites within the cell, and therefore regulatory information must exist in the structure of the protein to assure that they arrive at the correct destination. Our major objective is to identify this information, using as a model the 19 kDa plasma membrane glycoprotein (gp19K) coded by early region E3 adenovirus 1 (Ad2) or Ad5, and using a genetic approach. Gp19K is an abundant protein whose structure is known, and viable virus mutants can easily be constructed by in vitro site directed mutagenesis. Antisera are available against purified gp19K as well as synthetic peptides corresponding to gp19K. Gp19K has a 17 amino acid N-terminal "signal" sequence, two high-mannose oligosaccharides, and a 20 amino acid hydrophobic transmembrane domain followed by a 15 amino acid polar domain at the C-terminus. We have isolated virus mutants with sequenced lesions in the signal sequence, the glycosylation sites, and the C-terminal hydrophobic and/or polar domains. We propose to characterize these mutants in terms of processing and transport. Data are now available with some mutants. In a collaborative study, the mutants will be studied as a unique model for viral antigenclass 1 MHC antigen interaction. Other plasmid mutants, some sequenced, are available to construct additional virus mutants. We propose two novel and simple methods that apply standard techniques in bacterial genetics to isolate random missense mutations in gp19K. Region E3 encodes several other membrane proteins whose structures are fundamentally different from gp19K. We propose genetic studies, similar to those with gp19K, to identify the membrane-association and transport signals for these proteins. Virus mutants in some of the these genes, including fusions to gp19K, are currently available and additional mutants are easily constructed. Peptide antisera targeted to each of these proteins are available, and potent antisera directed fusion proteins synthesized by expression vectors will be generated.