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The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells.

ABSTRACT: The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-kappaB binding sites within the MIEP enhancer and cellular NF-kappaB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-kappaB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-kappaB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states.

SUBMITTER: Caposio P

PROVIDER: S-EPMC2863749 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells.

Caposio Patrizia P Luganini Anna A Bronzini Matteo M Landolfo Santo S Gribaudo Giorgio G

Journal of virology 20100210 9

The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate ...[more]

PMID: 20147408

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Project description:The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins.

Dataset Information

The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells.

Publications

The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells.

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