Efficiency and fidelity of full-site integration reactions using recombinant simian immunodeficiency virus integrase

Full-site integration by recombinant wild-type and mutant simian immunodefi ciency virus (SN) integrase (IN) was investigated with linear retrovirus-li ke DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a targe t substrate. Under optimized conditions, recombinant SIV IN produced donor- target products consistent with full-site (two donor ends) and half-site (o ne donor end) reactions with equivalent frequency. Restriction enzyme analy sis of the 3.8-kbp full-site reaction products confirmed the concerted inse rtion of two termini from separate donors into a single target molecule. Do nor ends carrying the viral U5 termini were preferred over U3 termini for p roducing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-ta rget junctions of the cloned products were characterized by sequencing. Ana lysis of 149 recombinants demonstrated approximately 84% fidelity for the a ppropriate simian retrovirus 5-bp host duplication. As seen previously in s imilar reactions with human immunodeficiency virus type 1 (HIV-1) IN from l ysed virions, approximately 8% of the donor-target recombinants generated w ith recombinant SN IN incurred specific 17- to 18- or 27- to 29-bp deletion s. The efficiency and fidelity of the full-site integration reaction mediat ed by the purified, recombinant SIV LN is comparable to that of HIV-1 IN fr om virions. These observations suggest that a purified recombinant lentivir us IN is itself sufficient to recapitulate the full-site integration proces s.