Efficient and tightly regulated expression systems for the human cytomegalovirus major transactivator protein IE2p86 in permissive cells.
The 86-kDa IE2 protein (IE2p86) of human cytomegalovirus is a pleiotropic regulatory polypeptide that is essential for activation of viral early promoters and thus, for the entire viral replication cycle. Moreover, this protein modulates cellular gene expression and contributes to the pathogenic features of HCMV. The full spectrum of IE2p86 mediated effects on cellular gene expression has not been defined yet, since efficient expression systems for this protein in HCMV permissive cells are, so far, limited. Here, we report the establishment of two efficient model systems that allow a tightly regulated expression of IE2p86 in various permissive cell types including primary human fibroblasts, primary endothelial cells and U373MG cells. Firstly, we generated a tetracycline-regulated U373MG cell line, which expresses high levels of IE2p86 upon tetracycline removal from the culture medium. Secondly, a recombinant baculovirus was constructed, which expresses IE2p86 under the control of the HCMV major immediate early enhancer/promoter upon transduction of various cell types. Importantly, IE2p86 was functional in both systems, since strong transactivation of luciferase promoter constructs could be measured. Furthermore, a cell cycle arrest was detectable after infection of primary human fibroblasts with IE2p86-expressing baculoviruses. Both expression systems represent useful tools to fully define the effects of this pleiotropic transactivator on cellular gene expression and to establish screening systems for novel antiviral drugs targeting this critical immediate early protein of HCMV.
Kronschnabl
Martina
M
Institut für Klinische und Molekulare Virologie, Schlossgarten 4, 91054 Erlangen, Germany.
Marschall
Manfred
M
Stamminger
Thomas
T
D000818:Animals; D016367:Baculoviridae; D002453:Cell Cycle; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014759:Viral Envelope Proteins; D014764:Viral Proteins
C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C077406:UL115 protein, Human herpesvirus 5; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C071440:glycoprotein H, Cytomegalovirus; C499358:glycoprotein H, Human cytomegalovirus; C439068:glycoprotein O, cytomegalovirus; D013752:Tetracycline