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Journal of Virology, June 2004, p. 6190-6199, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6190-6199.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Early Detection of a Two-Long-Terminal-Repeat Junction Molecule in the Cytoplasm of Recombinant Murine Leukemia Virus-Infected Cells

Fatima Serhan,1,{dagger} Magalie Penaud,1,{dagger} Caroline Petit,2 Thierry Leste-Lasserre,2 Stéphane Trajcevski,3 David Klatzmann,3 Ghislaine Duisit,1 Pierre Sonigo,2 and Philippe Moullier1*

INSERM U649, Nantes,1 INSERM U567, ICGM Cochin,2 CNRS/UPMC UMR 7087, Hôpital Pitié-Salpétrière, Paris, France3

Received 31 July 2003/ Accepted 16 March 2004

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-long-terminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.

* Corresponding author. Mailing address: INSERM ERM 0-105, CHU Hôtel-Dieu, 30, blvd. Jean Monnet, 44035 Nantes Cedex 01, France. Phone: (33) 240087490. Fax: (33) 240087491. E-mail: moullier{at}sante.univ-nantes.fr.

{dagger} F.S. and M.P. contributed equally to this work.

Journal of Virology, June 2004, p. 6190-6199, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6190-6199.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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