Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells.
An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.
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Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
van Gent
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Plasterk
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D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D002472:Cell Transformation, Viral; D004279:DNA, Viral / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D015498:HIV-2 / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D015345:Oligonucleotide Probes; D010957:Plasmids; D011533:Proviruses / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral