Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences

Abstract

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.