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Journal of Virology, April 1999, p. 2994-3003, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

HMG Protein Family Members Stimulate Human Immunodeficiency Virus Type 1 and Avian Sarcoma Virus Concerted DNA Integration In Vitro

Patrick Hindmarsh,1 Todd Ridky,1 Ray Reeves,2 Mark Andrake,3 Anna Marie Skalka,3 and Jonathan Leis1,*

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-49351; Washington State University, Pullman, Washington 991642; and Fox Chase Cancer Center, Philadelphia, Pennsylvania 191113

Received 13 August 1998/Accepted 21 December 1998

We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for avian sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571-3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.

* Corresponding author. Present address: Department of Microbiology, Northwestern University School of Medicine, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0338. FAX: (312) 503-1339. E-mail: j-leis{at}nwu.edu.

Journal of Virology, April 1999, p. 2994-3003, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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