Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions

Rodrigo A. Villanueva, Colleen Jonsson, Jennifer Jones, Millie M. Georgiadis, Monica J. Roth

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Retroviral 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.

Original languageEnglish (US)
Pages (from-to)146-160
Number of pages15
JournalVirology
Volume316
Issue number1
DOIs
StatePublished - Nov 10 2003

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Moloney murine leukemia virus
Integrases
Proteins
Viral DNA
Virus Replication
Recombinant Proteins
Oligonucleotides
Solubility
Gel Chromatography
Heparin
Chromatography
Genome

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. / Villanueva, Rodrigo A.; Jonsson, Colleen; Jones, Jennifer; Georgiadis, Millie M.; Roth, Monica J.

In: Virology, Vol. 316, No. 1, 10.11.2003, p. 146-160.

Research output: Contribution to journalArticle

Villanueva, Rodrigo A. ; Jonsson, Colleen ; Jones, Jennifer ; Georgiadis, Millie M. ; Roth, Monica J. / Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions. In: Virology. 2003 ; Vol. 316, No. 1. pp. 146-160.
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