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Journal of Virology, June 2002, p. 5540-5547, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5540-5547.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation and Analysis of Retroviral Integration Targets by Solo Long Terminal Repeat Inverse PCR

Yi Feng Jin,1 Toshio Ishibashi,2 Akio Nomoto,1 and Michiaki Masuda1,3*

Department of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033,1 Department of Otolaryngology, Social Insurance Central General Hospital, Tokyo 169-0073,2 Department of Microbiology, School of Medicine, Dokkyo University, Tochigi 321-0293, Japan3

Received 5 July 2001/ Accepted 4 February 2002

Upon retroviral infection, the genomic RNA is reverse transcribed to make proviral DNA, which is then integrated into the host chromosome. Although the viral elements required for successful integration have been extensively characterized, little is known about the host DNA structure constituting preferred targets for proviral integration. In order to elucidate the mechanism for the target selection, comparison of host DNA sequences at proviral integration sites may be useful. To achieve simultaneous analysis of the upstream and downstream host DNA sequences flanking each proviral integration site, a Moloney murine leukemia virus-based retroviral vector was designed so that its integrated provirus could be removed by Cre-loxP homologous recombination, leaving a solo long terminal repeat (LTR). Taking advantage of the solo LTR, inverse PCR was carried out to amplify both the upstream and downstream cellular flanking DNA. The method called solo LTR inverse PCR, or SLIP, proved useful for simultaneously cloning the upstream and downstream flanking sequences of individual proviral integration sites from the polyclonal population of cells harboring provirus at different chromosomal sites. By the SLIP method, nucleotide sequences corresponding to 38 independent proviral integration targets were determined and, interestingly, atypical virus-host DNA junction structures were found in more than 20% of the cases. Characterization of retroviral integration sites using the SLIP method may provide useful insights into the mechanism for proviral integration and its target selection.

* Corresponding author. Mailing address: Department of Microbiology, Dokkyo University School of Medicine, Tochigi 321-0293, Japan. Phone: 81-282-87-2131. Fax: 81-282-86-5616. E-mail: m-masuda{at}dokkyomed.ac.jp.

Journal of Virology, June 2002, p. 5540-5547, Vol. 76, No. 11
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.11.5540-5547.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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