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Journal of Virology, October 1999, p. 8104-8111, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Efficiency and Fidelity of Full-Site Integration Reactions Using Recombinant Simian Immunodeficiency Virus Integrase

Goodarz Goodarzi,1 Michael Pursley,1 Peter Felock,2 Marc Witmer,2 Daria Hazuda,2 Karl Brackmann,3 and Duane Grandgenett1,*

Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 631101; Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 194862; and Genetics Systems Corporation, Redmond, Washington 980523

Received 12 May 1999/Accepted 7 July 1999

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.

* Corresponding author. Mailing address: St. Louis University Health Sciences Center, Institute for Molecular Virology, 3681 Park Ave., St. Louis, MO 63110. Phone: (314) 577-8411. Fax: (314) 577-8406. E-mail: Grandgdp{at}SLU.EDU.

Journal of Virology, October 1999, p. 8104-8111, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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