The Cyclin E Promoter Is Activated by Human Cytomegalovirus 86-kDa Immediate Early Protein

Wade A Bresnahan,Thomas Albrecht,E Aubrey Thompson

doi:10.1074/jbc.273.34.22075

Wade A Bresnahan, Thomas Albrecht + Show 1 more

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https://doi.org/10.1074/jbc.273.34.22075

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Abstract

Human cytomegalovirus (HCMV) activates cyclin E/Cdk2, which regulates cell cycle progression in G1 and S phase of the cell cycle. HCMV activation of cyclin E/Cdk2 can be demonstrated in cells that are refractory to normal mitotic stimuli. This observation suggests that the virus has some means to overcome the stringent control on expression of cell cycle progression factors that is characteristic of cells in the G0 state. One of the mechanisms involved in activation of cyclin E/Cdk2 is the induction of cyclin E expression. We report here that HCMV induces cyclin E expression through a transcriptional mechanism. The cyclin E gene is activated by the HCMV 86-kDa immediate early gene product (IE86), which directly binds to nucleotide sequences within the cyclin E promoter. An IE86 DNA-binding mutant neither binds nor activates the cyclin E promoter. IE86-binding sites within the cyclin E promoter are required for IE86-mediated activation, and deletion of the IE86-binding site inhibits IE86 activation of the cyclin E promoter. We also demonstrate that mutation of the known E2F-binding sites in the cyclin E promoter does not block activation by HCMV or IE86. These data provide a molecular mechanism for HCMV induction of cyclin E and represent the first report of IE86 directly binding to a cellular promoter.

Highlights

Activation of cyclin-dependent kinase 2 (Cdk2)1 is essential for progression through the G1 phase of the cell cycle [1,2,3,4]
The kinetics of induction of cyclin E during human cytomegalovirus (HCMV) infection suggest that the products of one or more of the immediate early (IE) genes might play a role in cyclin E expression
HCMV immediate early expression plasmids were a generous gift from Dr Jay Nelson and have been described previously [26, 28]. pRLSV40-Luc was purchased from Promega. p86DEL-Luc was constructed by using oligonucleotides (5Ј-GGTACCCTGTCACTTGGCCCCGCC and 5ЈGAGCTCCTGCGCCCGGCTC) as PCR primers to amplify a fragment from Ϫ207 to ϩ35 from pE(Ϫ207)Luc

Summary

Introduction

Activation of cyclin-dependent kinase 2 (Cdk2) is essential for progression through the G1 phase of the cell cycle [1,2,3,4]. HCMV has the ability to promote exit from a G0 state, and some aspects of cell cycle progression can be demonstrated in HCMV-infected cells, which arrest in a state that resembles late G1 or early S phase [17,18,19] During this process, the virus affects a selective activation of cyclin E/Cdk2 [17, 20]. The mechanism by which immediate early gene products regulate transcription is not completely understood, there is increasing evidence that such factors act through direct DNA interaction and through protein-protein interactions with viral and cellular proteins. The kinetics of cyclin E induction, the ability of immediate early gene products to activate cellular promoters, and the ability of

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