We plan to identify genetic sequences of recombinant murine leukemia viruses (MuLV) which are required to induce each of three distinct stages of viral leukemogenesis which we have defined. We will study a system whereby events of leukemogenesis in AKR mice can be synchronized by intrathymic injection of cloned, recombinant MuLV. The role in the disease process of gp70, p15(E), and LTR regions of the viral genome will be tested by use of viruses constructed in vitro from subgenomic fragments of pathogenic and nonpathogenic MuLV. Virus-infected, preleukemic thymocytes will be characterized in three ways. Patterns of expression of MuLV antigens and differentiation alloantigens will be determined by quantitative immunofluorescence using multiparameter flow cytometric techniques. This is a powerful approach which will allow us to detect changes in minor populations of thymocytes. Fractionation of novel populations by cell sorting is also possible. Biochemical analysis of MuLV proteins in total and fractionated populations of infected thymocytes will determine whether metabolism of env gene products and their cellular localization correlate with the ability of a particular virus construct to induce premalignant changes. Functional assays will be developed to determine whether infection of thymocytes results in altered patterns of response to growth regulatory factors such as mitogenic interleukins, products of thymus epithelial cells, or transferrin.
