The aim of this proposal is to isolate and characterize a library of mutants of the Moloney and Abelson murine leukemia viruses. The mutations will define those regions in and near the Long Terminal Repeat (LTR) sequences of these viruses which are required for a variety of control functions: packaging of the viral RNA genome into virions; reverse transcription of the RNA into double-standard DNA; conversion of this DNA to the two closed circular viral DNAs; and especially integration of the DNA into the host chromosome. We have recently prepared full-length recombinant clones of the Moloney and Abelson virus genomes which are biologically active in transfection assays. We will therefore be able to alter these DNAs by site-specific mutagenesis, directing the mutations to the LTRs and to neighboring sequences involved in control functions. Both deletion and base-change mutations will be introduced into the cloned DNA by combinations of enzymatic and chemical means. The altered DNAs will then be introduced into murine cells by transfection and stocks of virus (which carry the mutation) prepared from these cells. This protocol will allow the isolation of mutants which are replication-defective. Transfection of viral DNA bypasses the early steps of infection and thus allows the mutated DNA to produce virus. The virus will be used to infect new cells, and the course of this infection analyzed biochemically to determine the stage blocked by the mutation. These mutants will help us understand in detail several of the early events in the life cycle of the retroviruses, as well as the mechanisms used by the host cells to carry out the related process of DNA transposition. This information will be of great significance as part of our knowledge about normal cellular genetic processes and about how retroviruses affect them to cause leukemia and sarcomas. In addition, the mutants will make possible future studies on viral recombination and on the use of retrovirus genomes as vectors for the transfer of cellular genes from cell to cell.
NIH ExPorter
NIH ExPorter