Experiments are proposed to study the role of the retrovirus encoded endonuclease in retroviral DNA integration and the role of host cell genes in retrovirus replication. The planned strategies will exploit the experimental advantages of the Moloney murine leukemia virus (Mo-MLV) system, using modern genetic and biochemical techniques. A series of defined point mutations, frameshift mutations and in-frame deletions will be introduced into the Mo-MLV endonuclease coding region by in vitro mutagenesis techniques. Virions derived from the mutagenized DNAs will be analysed for the ability to efficiently integrate metabolic marker pseudotypes. The integrated marker proviruses contain additional sequences which allow them to be amplified (along with adjacent host DNA) in mammalian cells and rapidly isolated as plasmids in bacterial cells. Sequencing of mutant generated proviral junctions may allow the identification of the endonuclease domains responsible for one or more of the precise DNA cleavage steps in the integration process. Expression of the MLV endonuclease independent of other viral proteins will be attempted in mouse cells so as to determine whether the integration function can be supplied in trans to integration defective viruses and viral DNA. The production of the MLV endonuclease will also be attempted in two different bacterial expression systems. If significant amounts of biologically active protein is synthesized, biochemical analyses and comparisons of purified wild type and mutant endonucleases will be attempted. It may be possible to correlate integration-defectiveness in vivo to specific mutant endonuclease biochemical deficiencies in vitro. Finally, experiments are proposed to identify and characterize mouse cellular genes which are required for retrovirus replication. Mouse cells will be mutagenized and cells incapable of replicating a marker virus will be selected. The resulting host mutants will be examined and characterized for the nature of their viral replication block. The isolation of selected host mutant genes will be initiated.
| Donehower, L A (1988) Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol. J Virol 62:3958-64 |
