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
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.