The overall goal of this competitive renewal application is to continue our work on the cellular and molecular characterization of Mls-1, the prototype of the endogenous superantigens (SAGs) which is encoded by the endogenous Murine Mammary Tumor Virus (MMTV) Mtv-7. Furthermore, our study of virally encoded SAGs will be expanded by testing the human pathogens EBV and HIV-1 for this gene trait. The following specific aims are proposed: A. Because SAG expression is critical for successful transmission of infectious MMTV, it is important to understand the control of expression of this molecule. Our preliminary data that MMTV sag transcription is controlled independently of that of the other MMTV genes will be continued. Using transient expression of a reporter construct as read-out, the MMTVenv gene will be tested for promoter/enhancer elements, the octamer sites in the 5' and 3' LTR for B cell specific expression, and the role of the glucocorticoid responsive elements in Mls-1 expression. B. Since it is not known how and when Mls-1 associates with MHC class II molecules, its post- translational pathway will be analyzed by transfecting it into cells that have a general defect in processing class II restricted antigens, as well as cells that vary in their level and form of MHC class II Ii chain expression. Many retroviral proteins, including HIV-1 gp160 and MMTV env, require processing by furin to be functional. Since MMTV SAGs have two potential furin cleavage sites which are highly conserved, the role of this protease for functional Mls-1 expression will be tested by over-expression of furin, as well as by transient expression of a furin inhibitory protein. C. The apparent tri-molecular interaction of Mls-1, MHC class II and the TCR will be analyzed in vitro. Soluble recombinant Mtv-7 sag products and mutants thereof, as well as soluble recombinant TCRs and mutants thereof will be produced. These recombinant products will be tested in functional assays, and the affinity of their interaction with soluble MHC class II molecules will be measured with the help of a Biosensor. D. The human herpesvirus EBV is the causative agent of infectious mononucleosis, which is characterized by a strong self-limited T cell proliferation. In order to investigate whether this is due to a SAG-like response, EBV will be tested for SAG expression. An autologous system will be established for screening wildtype EBV, derived from mononucleosis patients, for T cell proliferation. The Vbeta profile of the responding T cells will be compared to that expressed after mitogen stimulation. If positive results are obtained, well characterized EBV recombinants will be used for mapping the gene responsible for the SAG activity. Finally, the hypothesis will be tested that HIV-1 makes use of a SAG for facilitating viral transmission. Since our laboratory has shown that human T cells respond in a Vbeta specific fashion to the murine retroviral SAG Mls-1, murine T cells will be tested for a Vbeta specific proliferation in response to HIV-1 gene products. The feasibility of all these approaches has already been established, and preliminary data have been collected for the studies involving human pathogens. New insights may be gained for the development of therapeutics and prevention of viral infection.
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