STRUCTURAL AND FUNCTIONAL ANALYSIS OF ENDOGENOUS PROVIRUSES OF MICE

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Mouse genome contains at least 50 copies of murine leukemia viral (MuLV)-related DNAs, the majority of which are defective. Recombination between sequences of different infectious and defective MuLV DNAs results in the generation of novel MuLVs including lymphomagenic mink cell focus-forming (MCF) viruses. The main goals of this project are: 1) to determine the relatedness between endogenous MuLV sequences and infectious MuLVs; 2) to study generation of recombinant MCF viruses; and 3) to identify sequences contributing to leukemogenicity of MCF MuLVs. Two unique retroviral DNAs, B-26 and B-60, were cloned from the BALB/c mouse genome. The DNAs were distantly related to each other and known infectious MuLV proviruses in the gag, pol and env regions. Nucleotide sequence analysis indicated that B-26 and B-60 DNAs contained different retroviral-like LTRs, distinct from MuLV LTRs. Comparative distribution studies of B-26- and B-60-related sequences and infectious MuLV proviruses in a series of rodent DNAs indicated that B-26 and B-60 gene families were ancient and had amplified in the mouse genome. To study the structure of potential recombinational partners in MCF virus genesis, a 2.0 kb cDNA segment of endogenous MCF env-related 7.2 kb mRNA species was cloned and its nucleotide sequence determined. Sequence analysis indicated a 1.2 kb deletion in the env region. The cDNA was found to be closely related to a previously cloned endogenous MCF-related provirus which contained an identical env deletion, thus indicating that the latter DNA family might serve as templates to possible precursors of MCF viruses. Leukemogenic MCF MuLVs contain a unique 12 bp sequence in the integrase-coding region of the pol gene. To study the role of this nucleotide stretch in leukemogenesis the 12 bp were deleted by oligonucleotide mutagenesis from MCF13 proviral DNA. The virus obtained from transfection studies using the mutant DNA has been injected into young AKR mice to test whether the MCF viral genome retains its leukemogenic potential in the absence of the 12 bp sequence.