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TheMov-7 andMov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at theMov-7 locus andMov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying theMov-9 locus but is not activated in animals carrying theMov-7 locus. Consequently, onlyMov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to theMov-7 andMov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that theMov-7 andMov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10−5 XC plaques per genome equivalent were induced by theMov-9 clone, whereas only 10−9 XC plaques per genome equivalent were induced by theMov-7 clone. Moreover, NIH 3T3 cells transfected with theMov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with theMov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at theMov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression inMov-7 andMov-9 mice correlates with the biological properties of the respective clones. Genomic DNA fromMov-9 mice was not infectious in the transfection assay (specific infectivity &lt; 10−7 PFU per genome equivalent). As the only difference between the genomic and the clonedMov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.

journal of virology

Cloning of Two Genetically Transmitted Moloney Leukemia Proviral Genomes: Correlation Between Biological Activity of the Cloned DNA and Viral Genome Activation in the Animal