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Bcl-3-Regulated Transcription from Major Immediate-Early Promoter of Human Cytomegalovirus in Monocyte-Derived Macrophages¹

Kashif Aziz Khan^*, Alain Coaquette^*, Christian Davrinche and Georges Herbein^2,*

^* Department of Virology, Institut Fédératif de Recherche 133, Equipe d’Accueil 3186, Franche-Comté University, Centre Hospitalier Universitaire de Besançon, Besançon, France; and Institut National de la Santé et de la Recherche Médicale, Unité 563, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France

Monocytes/macrophages are key cells in the pathogenesis of human CMV (HCMV) infection, but the in vitro rate of viral production in primary human monocyte-derived macrophages (MDM) is considerably lower than in fibroblasts. Considering that the NF-B signaling pathway is potentially involved in the replication strategy of HCMV through efficient transactivation of the major immediate-early promoter (MIEP), efficient viral replication, and late gene expression, we investigated the composition of the NF-B complex in HCMV-infected MDMs and fibroblasts. Preliminary studies showed that HCMV could grow in primary MDM culture but that the viral titer in culture supernatants was lower than that observed in the supernatants of more permissive MRC5 fibroblasts. EMSA and microwell colorimetric NF-B assay demonstrated that HCMV infection of MDMs increased p52 binding activity without activating the canonical p50/p65 complex. Moreover, Bcl-3 was up-regulated and was demonstrated to associate with p52, indicating p52/Bcl-3 complexes as the major component of the NF-B complex in MDMs. Luciferase assays in promonocytic U937 cells transfected with an MIEP-luciferase reporter construct demonstrated MIEP activation in response to p52 and Bcl-3 overexpression. Chromatin immunoprecipitation assay demonstrated that p52 and Bcl-3 bind the MIEP in acutely HCMV-infected MDMs. In contrast, HCMV infection of MRC5 fibroblasts resulted in activation of p50/p65 heterodimers. Thus, activation of p52/Bcl-3 complexes in MDMs and p50/p65 heterodimers in fibroblasts in response to HCMV infection might explain the low-level growth of the virus in MDMs vs efficient growth in fibroblasts.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants from Franche-Comté University (to G.H.). K.A.K. is a recipient of a doctoral scholarship from the Higher Education Commission, Pakistan. Results of these studies were presented in part at the 11th International CMV and Beta Herpesvirus Workshop, Toulouse France, May 13–17, 2007.

² Address correspondence and reprint requests to Dr. Georges Herbein, Department of Virology, Franche-Comté University, Hôpital Saint-Jacques, 2, place Saint-Jacques, F-25030 Besançon cedex, France. E-mail address: gherbein{at}chu-besancon.fr

³ Abbreviations used in this paper: HCMV, human CMV; IKK, IB kinase; AD169, high-passage laboratory strain AD169; ChIP, chromatin immunoprecipitation; MDM, primary human monocyte-derived macrophage; MIEP, major immediate-early promoter; MOI, multiplicity of infection; siRNA, small interfering RNA; EBNA, Epstein-Barr nuclear Ag; HDAC, histone deacetylase.