DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro

Citation
A. Vora et Dp. Grandgenett, DNase protection analysis of retrovirus integrase at the viral DNA ends for full-site integration in vitro, J VIROLOGY, 75(8), 2001, pp. 3556-3567
Citations number
44
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF VIROLOGY
ISSN journal
0022-538X → ACNP
Volume
75
Issue
8
Year of publication
2001
Pages
3556 - 3567
Database
ISI
SICI code
0022-538X(200104)75:8<3556:DPAORI>2.0.ZU;2-Z
Abstract
Retrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating t he DNA termini in a concerted fashion into exogenous target DNA (full site) , mimicking integration in vivo. Molecular insights into the organization o f avian myeloblastosis virus IN at the viral DNA ends were gained by recons tituting nucleoprotein complexes possessing intasome characteristics. Assem bly of IN-4.5-kbp donor complexes capable of efficient full-site integratio n appears cooperative and is dependent on time, temperature, and protein co ncentration. DNase I footprint analysis of assembled IN-donor complexes cap able of full-site integration shows that wild-type U3 and other donors cont aining gain-of-function attachment site sequences are specifically protecte d by IN at low concentrations (<20 nM) with a defined outer boundary mappin g <similar to>20 nucleotides from the ends, A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DN ase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 end s for full-site integration shows binding by IN at low concentrations proba bly occurs only at the very terminal nucleotides (<10 bp) on U5. The result s suggest that assembly requires a defined number of avian IN subunits at e ach viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.