Retroviruses have provided great insights into the process of carcinogenesis. In the past, studies on retroviral carcinogenesis have elucidated such important principles as viral oncogenes and insertional activation of cellular proto-oncogenes. Retroviruses are also useful for studying the multi-step process of carcinogenesis, since they induce specific tumors with predictable latencies. Moloney murine leukemia, virus (M-MuLV) induces T-lymphoma with a latency of 3-4 months when injected into mice. In this proposal, we will continue our experiments on mechanism of leukemogenesis by M-MuLV. We will address three aims. 1) We will continue to map the initial cellular pathways of infection, from the initially infected cells through the bone marrow to the thymus. We will take advantage of replication-defective, helper-free M-MuLV-based vectors expressing a readily detectable reporter gene (beta-gal). Infection of mice mutant in development of myeloid cells or hair follicles, and adoptive transfer experiments will address the mechanisms of the spread of infection. 2) We will investigate the role of glycosylated gag protein in M-MuLV infection in vivo. We previously studied the synthesis of glycosylated gag protein, and generated a glycosylated gag-negative M-MuLV mutant (Ab-X-M-MuLV). In vivo infections with this mutant indicate that glycosylated gag is important for M-MuLV infection in vivo. Moreover, SC inoculation of the mutant shows a delay in leukemogenesis, and the tumors show lower levels of infectivity. We will employ genetic and biochemical means to study the role of glycosylated gag in M-MuLV infection in vivo. 3) We will study the basis of tissue-specific expression of the M-MuLV long terminal repeat (LTR), the major determinant in MuLV disease specificity. Other investigators have extensively mapped nuclear factor binding sites within the M-MuLV, but it has been unclear if those proteins are bound in vivo. We have applied in vivo footprinting and ligation-mediated PCR (LMPCR) to map factor binding to the M-MuLV LTR in infected cells. We will now map factor binding in the upstream and downstream LTRs, and we will characterize factor binding to wild-type or interesting variant LTRs in different cell types where there is high-level vs. low expression. Together, these experiments will provide new and important insights into leukemogenesis by MuLVs.
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