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Journal of Virology, February 2001, p. 1132-1141, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1132-1141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Base-Pair Substitutions in Avian Sarcoma Virus U5 and U3 Long Terminal Repeat Sequences Alter the Process of DNA Integration In Vitro

Patrick Hindmarsh,1 Michael Johnson,1 Ray Reeves,2 and Jonathan Leis3,*

Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-49351; Washington State University, Pullman, Washington 991642; and Department of Microbiology and Immunology, Northwestern University School of Medicine, Chicago, Illinois 606113

Received 17 August 2000/Accepted 25 October 2000

We have described a reconstituted avian sarcoma virus (ASV) concerted DNA integration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human high-mobility-group protein I(Y). Integration in this system is dependent upon the mini-donor DNA having IN recognition sequences at both ends and upon both ends of the same donor integrating into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated from bacteria containing drug-resistant markers with amber mutations. This system was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserved CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integrants from reactions with mutated U5 but not U3 IN recognition sequences were products that contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the process of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonconcerted DNA integration are more common in U5 than in U3.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Northwestern University School of Medicine, 303 Chicago Ave., Chicago, IL 60611. Phone: (312) 503-1166. Fax: (312) 503-1339. E-mail: j-leis{at}northwestern.edu.

Journal of Virology, February 2001, p. 1132-1141, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1132-1141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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