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J. Virol. doi:10.1128/JVI.02015-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Ku80 participates to the targeting of retroviral transgenes in chromatin of CHO cells

LBPA, CNRS, E.N.S. Cachan, 61 avenue du Président Wilson, 94235 Cachan, France; Régulation des Infections Rétrovirales, Institut Pasteur, 75015 Paris; Laboratoire d'Analyse d'images en pathologie cellulaire, Institut Universitaire d'Hématologie, Hôpital St louis, 1 avenue Vellefaux 75010 Paris, Laboratoire d'Andrologie, CHU Bicêtre, 94270 Kremlin Bicêtre

^* To whom correspondence should be addressed. Email: mouscadet{at}lbpa.ens-cachan.fr.

	Abstract

The heterodimer Ku70/80 Ku is the DNA binding component of the DNA-PK complex required for the non-homologous end-joining pathway (NHEJ). It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-LTR viral DNA circles, viral DNA integration and transcription. Here, we describe the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps by demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analysed the transgene distributions localisation in nucleus by applying a 3D-reconstruction model to 2D FISH images. This indicated that the presence of Ku80 resulted in a bias towards the transgenes being located at the periphery of the nucleus associated with their being repressed; in absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not being strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.