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Journal of Virology, October 2003, p. 11060-11071, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11060-11071.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Junonia coenia Densovirus-Based Vectors for Stable Transgene Expression in Sf9 Cells: Influence of the Densovirus Sequences on Genomic Integration

Hervé Bossin,1 Philippe Fournier,1 Corinne Royer,2 Patrick Barry,1 Pierre Cérutti,1 Sylvie Gimenez,1 Pierre Couble,3 and Max Bergoin1*

Unité de Virologie Moléculaire, UMR5087, INRA-CNRS-UMII, Station de Recherches de Pathologie Comparée, 30380 Saint-Christol-les-Alès, and Laboratoire de Pathologie Comparée, Université Montpellier II,1 Unité Nationale Séricicole, INRA, 69350 La Mulatière,2 Centre de Génétique Moléculaire & Cellulaire, CNRS UMR 5534, 69622 Villeurbanne Cedex, France3

Received 8 April 2003/ Accepted 23 July 2003

The invertebrate parvovirus Junonia coenia densovirus (JcDNV) shares similarities with terminal hairpins and nonstructural (NS) protein activities of adeno-associated virus (AAV) despite their evolutionary divergence (B. Dumas, M. Jourdan, A. M. Pascaud, and M. Bergoin, Virology, 191:202-222, 1992, and C. Ding, M. Urabe, M. Bergoin, and R. M. Kotin, J. Virol. 76:338-345, 2002). We demonstrate here that persistent transgene expression in insect cells results from stable integration of transfected JcDNV-derived vectors into the host genome. To assess the integrative properties of JcDNV vectors, the green fluorescent protein (GFP) gfp marker gene was fused in frame into the major open reading frame (ORF1) of the viral sequence under the control of the P9 capsid protein promoter. In addition, the influence of the nonstructural proteins on the posttransfection maintenance of the vectors was examined by interruption of one or all three NS ORFs. Following transfection of Sf9 cells with each of the JcDNV constructs, clones showing persistent GFP expression were isolated. Structural analyses revealed that the majority of the JcDNV plasmid sequence was integrated into the genome of the fluorescent clones. Integration was observed whether or not NS proteins were expressed. However, the presence of NS genes in the constructs greatly influenced the number of integrated copies and their distribution in the host genome. Disruption of NS genes expression resulted in integration of head-to-tail concatemers at multiple sites within the genome. Further analyses demonstrated that the cis JcDNV 5' inverted terminal repeat region was the primary site of recombination. Sequence analyses of integration junctions showed rearrangements of both flanking and internal sequences for most integrations. These findings demonstrate that JcDNV vectors integrate into insect cells in a manner similar to AAV plasmids in mammalian cells.

* Corresponding author. Mailing address: Université de Montpellier II, Laboratoire de Pathologie Comparée, CC 101 - Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. Phone: (33) 467 143 220. Fax: (33) 467 144 679. E-mail: bergoin{at}ensam.inra.fr.

Journal of Virology, October 2003, p. 11060-11071, Vol. 77, No. 20
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.20.11060-11071.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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