In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins

• Published in: Molecular and cellular biology Vol. 13; no. 2; pp. 1238 - 1250
• Main Authors: Klucher, K M, Sommer, M, Kadonaga, J T, Spector, D H
• Format: Journal Article
• Language: English
• Published: 01.01.1992
• ISSN: 0270-7306

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa Cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli -synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, The HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein.