Adenoviruses are small, non-enveloped viruses containing a linear double stranded DNA genome that were first discovered in 1945. The human adenoviruses are associated with a variety of diseases including upper respiratory infections, gastrointestinal illness, and conjunctivitis. For many years, these viruses have been outstanding model systems for the study of DNA replication, RNA synthesis, protein translation, oncogenic transformation, and apoptosis. Most recently, interest in adenovirus has expanded due to its potential as a vector for vaccination and human gene transfer studies. Viral gene expression, genome replication, and viral assembly all take place in the nucleus of the infected cell. This project focuses on assembly and, in particular, the encapsidation of viral DNA. This process requires at least three elements: the viral packaging domain, which is located near the left end of the genome and is made up of repeated elements called A repeats; the viral IVa2 protein, which binds to required sequence motifs in this region; and the viral 52/55 kDa protein, which binds the IVa2 protein and plays an as of yet undetermined role. The goals of this proposal are to elucidate further the mechanism by which adenovirus encapsidates its DNA. The role of the 52/55 kDa protein will be uncovered through the analysis of a mutant virus that doesn't express the protein and by structure-function studies on the protein. The mechanism by which the lVa2 protein facilitates DNA packaging will be determined by construction and characterization of a mutant virus that does not express the protein, by determining whether the protein has a predicted ATPase activity, and by examining functional interactions of the IVa2 protein with other viral proteins. Finally, an in vitro system for encapsidation of viral DNA will be developed in order to obtain a detailed understanding of the roles of these two proteins, as well as other factors, in the process. The ultimate goal of the project is the development of an in vitro system for complete viral assembly. These studies will advance our basic understanding of how adenovirus packages its DNA and produces infectious virions. This knowledge will be applicable to the development of anti-viral drugs that block this process, as well as to the development of safer adenovirus vectors.
