This proposal outlines plans to characterize the transcriptional control machinery utilized by Moloney murine sarcoma virus (MSV) when it infects cultured mammalian cells. The long terminal repeat (LTR) of MSV which directs transcription of viral genes appears to be receptive to both positive and negative regulation in animal cells. My goals are to delineate the DNA sequences of the LTR that facilitate its activation and repression and identify the cellular proteins that mediate these processes. First, deletion mutants of the LTR will be constructed and assayed to establish the boundaries of the LTR transcriptional control sequences. Next, cellular proteins will be identified that bind in a sequence-specific manner within the boundaries of the transcriptional control signals. Then a more refined mutagenesis approach will be initiated in which base substitution mutations are introduced into protein binding sites such that binding is disrupted. Mutant LTR's will be assayed for transcriptional activity in four cell types. Mouse L cells will be employed to study the positive regulation of LTR expression. The mouse embryonal carcinoma F9 cell line provides an environment in which the LTR is negatively regulated. Frog oocytes also provide an environment to potentially study repression of LTR transcriptional activity. In rat XC cells, the role of proteins in the stimulation of MSV LTR transcription by glucocorticoids can be examined. After expression assays have indicated a role for specific proteins in either LTR repression or stimulation, double mutants (LTR's bearing two mutant binding sites) will be assayed to test whether protein factors act independently, synergistically or antagonistically. On the basis of the results of in vivo expression assays, in vitro binding studies will be performed to test directly for cooperative or antagonistic interactions between proteins. The resolution of the molecular mechanism by which the MSV LTR is positively and negatively regulated is of medical importance for two reasons. Murine retroviral expression vectors are being used in human gene therapy experiments. Fundamental information about LTR transcriptional control will aid researchers attempting to optimize expression of therapeutic genes being driven by retroviral control signals. An understanding of the transcriptional control of retroviruses may also contribute to the solution of the health problems posed by disease-causing retroviruses, such as the AIDS etiological agent HTLV-III.
