Molecular Cloning of B- and N-Tropic Endogenous BALB/c Murine Leukemia Virus Circular DNA Intermediates: Isolation and Characterization of Infectious Recombinant Clones

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The unintegrated closed circular DNA intermediates from B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were isolated from acutely infected mouse fibroblasts and cleaved at their unique HindIII sites. The linearized B- and N-tropic MuLV DNAs were then inserted in λ vector Charon 21A and cloned. Seventeen B-tropic and ten N-tropic recombinants were analyzed. The viral insert of these 27 MuLV recombinants could be grouped into 3 classes. The first class included permuted full-length molecules of 8.8 kilobase pairs (kbp) having two long terminal repeats (LTR) (0.55 kbp) mixed with 8.25-kbp molecules. The second class included 8.25-kbp molecules which harbored only one LTR copy. The third class of inserts was made of molecules with deletions of various lengths (0.1 to 3 kbp). These deletions were localized by restriction enzyme analysis. Significantly more deletions were observed in N-tropic than in B-tropic recombinants, although these two genomes isolated from acutely infected cells seemed to differ only by the absence of a 70-base-pair (bp) sequence in the N-tropic LTR. The biological activity of each of the recombinants of the first and second classes was assayed by transfection on NIH/3T3 cells. Two recombinants, one B-tropic (λ B-16) and one N-tropic (λN-20) were found to be infectious. Both were able to give rise to replication-competent MuLV which could form large XC plaques and had kept the tropism of its progenitor. Like its progenitor in vivo DNA, λN-20-cloned DNA lacked a 70-bp sequence in its unique LTR. The emerging N-tropic MuLV after transfection with λN-20 was used to acutely infect NIH/3T3 cells. The viral DNA intermediates isolated were now found to have two LTR copies. They had also acquired a 70-bp sequence in each LTR. The acquisition or loss of this 70-bp sequence did not seem to affect the replication, the XC plaque formation, or the tropism of the virus.

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