The focus of this proposal is the Sag (super-antigen) or orf gene of murine mammary tumor virus (MMTV). Recent experiments indicate that this protein, which is encoded in the MMTV LTR, functions as a super-antigen in infected mice. This leads to deletion of whole subsets of TCR-beta-specific T- lymphocytes. The Sag genes of endogenous MMTV proviruses have been found to be the same as the murine minor lymphocyte stimulating (Mls) antigens. The role of the Sag gene in the MMTV life cycle will be explored in these experiments. In recently published work, the investigator and co-investigator have shown that expression of an exogenous MMTV Sag in transgenic mice renders them resistant to infection by the same virus but not other MMTVs. This implicates infection of T- lymphocytes in the pathway of virus spread from the gut to the mammary gland. Recent results indicate that expression of Sag protein alone delays, but does not inhibit tumorigenesis in transgenic animals. In addition, lymphocytes from Sag transgenic animals do not display Mls activity in in vitro assays. On the other hand, animals transgenic for an entire MMTV provirus are much more resistant to tumorigenesis, and lymphocytes from these animals show Mls activity. This leads the investigators to hypothesize that other viral genes, (env in particular) are important for full superantigen function. A comprehensive series of experiments are proposed, with four major aims.
The first aim i nvolves molecular manipulations of the Sag gene. Frame shift and deletion mutations of Sag will be tested in transgenic mice to identify domains of the protein necessary for superantigen function and blockage of viral infection. In addition, chimeric Sag molecules will be generated to determine the region of the protein involved in TCR V-beta specificity, and Sag mRNA will be cloned and characterized.
The second aim will study the subcellular localization and tissue distribution of Sag. In order to do this, antigenically tagged Sag protein will be generated, and Sag-specific immunological reagents will be developed. The subcellular localization will be studied by immunoprecipitation, and tissue distribution will be studied by sensitive immunological techniques such as immuno-PCR. In the third aim, the role of Sag in the MMTV life cycle will be investigated. In particular, different lymphoid populations will be tested for the presence of infectious virus, and whether they can transfer viral infection to mammary gland cells. The effect of Sag gene mutations on viral infectivity will be tested, and infection in mutant mice lacking different lymphoid components will be tested. In the final aim, the role of MMTV envelope in Sag gene function and Mls response will be investigated.This will be accomplished by co-expressing env and Sag in cultured cells or transgenic mice, and testing whether the env sequences and sag sequences can cooperate in cis or trans.
| Wrona, T J; Lozano, M; Binhazim, A A et al. (1998) Mutational and functional analysis of the C-terminal region of the C3H mouse mammary tumor virus superantigen. J Virol 72:4746-55 |
