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Journal of Virology, March 2004, p. 2906-2920, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2906-2920.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Unintegrated Lentivirus DNA Persistence and Accessibility to Expression in Nondividing Cells: Analysis with Class I Integrase Mutants

Dyana T. Saenz,1,2 Nils Loewen,1 Mary Peretz,1 Todd Whitwam,1,2 Román Barraza,1 Kyle G. Howell,3 Jonathan M. Holmes,3 Margaret Good,3 and Eric M. Poeschla1,2,4*

Molecular Medicine Program,1 Departments of Immunology,2 Medicine,4 Ophthalmology, Mayo Clinic College of Medicine, Rochester, Minnesota 559053

Received 3 September 2003/ Accepted 8 November 2003

The circumstances under which unintegrated lentivirus DNA can persist and be a functional template for transcription and protein expression are not clear. We constructed and validated the first class I (nonpleiotropic) integrase (IN) mutants for a non-human lentivirus (feline immunodeficiency virus [FIV]) and analyzed both these and known class I human immunodeficiency virus type 1 IN mutants. The FIV IN mutants (D66V and D66V/D118A) had class I properties: Gag/Pol precursor expression, proteolytic processing, particle formation, and reverse transcriptase (RT) production were normal, while the transduction of dividing fibroblasts was prevented and integration was blocked. When injected into rat retinas, the wild-type (WT) vector produced extensive, persistent transgene expression, compared with only rare positive neuronal cells for the IN mutant vector. In contrast, both WT and mutant vectors produced entirely equivalent, effective transduction levels of primary rat neurons (retinal ganglion cells). By testing the hypothesis that the unexpected retinal neuron transduction was related to cell cycle status, we found that when fibroblasts were growth arrested, transduction and internally promoted transgene expression were not inhibited at all by the class I FIV or HIV-1 IN mutations. Cells were then transduced under aphidicolin arrest and were released from the block 48 h later. Vector expression was stable and durable during repeated passaging in WT vector-transduced cells, while the release of cells transduced with equivalent RT units of class I IN mutant FIV or HIV vector resulted in a steady decline of expression, from 97 to 0% of cells by day 10. Southern blot and PCR analyses showed a lack of integration, irrespective of cell cycle, for the class I mutants and an increase in one- and two-long terminal repeat circular and linear unintegrated DNAs in growth-arrested cells. We conclude that if cell division is prevented, unintegrated FIV and HIV-1 vector DNAs can produce high-level internally promoted transgene expression equivalent to WT vectors. The expression correlates with the unintegrated DNA levels. These observations may facilitate the study of the roles of IN and other preintegration complex components in preintegration phases of infection by (i) providing an alternative way to monitor unintegrated nuclear cDNA forms, (ii) restricting ascertainment to the transcriptionally functional subset of unintegrated DNA, (iii) enabling analysis in individual, nondividing cells, and (iv) uncoupling other potential functions of IN from integration.

* Corresponding author. Mailing address: Molecular Medicine Program, Guggenheim 18, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. Phone: (507) 284-3178. Fax: (507) 266-2122. E-mail: emp{at}mayo.edu.

Journal of Virology, March 2004, p. 2906-2920, Vol. 78, No. 6
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.6.2906-2920.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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