Efficiency and fidelity of full-site integration reactions using recombinant simian immunodeficiency virus integrase.
Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SIV) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SIV IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV IN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.
Goodarzi
G
G
Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
Pursley
M
M
Felock
P
P
Witmer
M
M
Hazuda
D
D
Brackmann
K
K
Grandgenett
D
D
D004926:Escherichia coli; D006801:Humans; D019426:Integrases / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D015302:Simian Immunodeficiency Virus / Q000502:physiology*; D013379:Substrate Specificity; D016662:Virus Integration / Q000235:genetics*
D011994:Recombinant Proteins; D019426:Integrases