Villanueva, Rodrigo A.Jonsson, Colleen B.Jones, JenniferGeorgiadis, Millie M.Roth, Monica J.2018-03-202018-03-202003-11-10Villanueva, R. A., Jonsson, C. B., Jones, J., Georgiadis, M. M., & Roth, M. J. (2003). Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. Virology, 316(1), 146–160.0042-6822https://hdl.handle.net/1805/15669Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni2+-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.en-USPublisher PolicyRetroviral integrasesviral DNAArticle