Footprints on the Viral DNA Ends in Moloney Murine Leukemia Virus Preintegration Complexes Reflect a Specific Association with Integrase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 18; pp. 10535 - 10540
• Main Authors: Wei, Shui-Qing, Mizuuchi, Kiyoshi, Craigie, Robert
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 01.09.1998; National Acad Sciences; National Academy of Sciences
• Subjects: 3T3 Cells; Animals; Base Sequence; Biochemistry; Biological Sciences; Deoxyribonucleic acid; DNA; DNA Footprinting; DNA, Viral; Genetic mutation; Integrases - metabolism; Integration host factors; Leukemia Virus, Murine - enzymology; Leukemia Virus, Murine - genetics; Mice; Moloney murine leukemia virus; Moloney virus; Murine leukemia virus; Mutagenesis, Site-Directed; NIH 3T3 cells; Nucleolus organizer region; Nucleoproteins; Plasmids; Polymerase Chain Reaction; Product category rules; Viral DNA; Virus Integration; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.18.10535

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3′end processing.