| Retroviruses have highly organized genomes that encode a large number of proteins from a relatively short nucleic acid sequence. They utilize a variety of methods to achieve this, including the use of overlapping reading frames and the production of multiple RNAs through alternative splicing. In order to efficiently express RNAs encoding structural and enzymatic proteins, complex retroviruses like human T-cell leukemia virus (HTLV) must first express a doubly spliced RNA encoding both the Tax and Rex positive transregulatory proteins in separate but partially overlapping reading frames. As a result, functional studies of each of these proteins in the context of replicating virus, particularly mutational studies, present unique challenges. This dissertation both describes and utilizes experimental systems for the study of Tax and Rex that model in vivo in the context of replicating virus in human T cells, the natural target of HTLV infection.; The Rex protein is required for the expression of singly spliced and unspliced viral RNAs that encode HTLV structural and enzymatic proteins. Prior to the publication of the second chapter of this dissertation, our knowledge of Rex function was based on studies using cDNA overexpression vectors and reporter assays conducted in non-human cell lines. In keeping with our desire to obtain data from systems that closely approximate natural infection, the work in chapter two of this dissertation used full-length proviral clones and human T cell lines to investigate the mechanism for the function of HTLV-2 Rex.; The third chapter of this dissertation presents tissue culture and molecular biology techniques developed to generate and characterize stably transfected producer cells that express HTLV-1 from a proviral clone.; The fourth chapter of this dissertation describes the construction of a chimeric proviral clone of HTLV-1 in which the Tax-response elements of the viral promoter are replaced with the cytomegalovirus immediate-early enhancer, to theoretically eliminate the requirement for Tax in efficient viral transcription and replication.; The last chapter of this dissertation describes a number of HTLV-1 Tax mutants that have been well characterized in in vitro systems for their ability to alter the activity of a variety of cellular pathways, many of which may be relevant to the oncogenic process in vivo. (Abstract shortened by UMI.)... |
