Regulation of the Transcription and Replication Cycle of Human Cytomegalovirus Is Insensitive to Genetic Elimination of the Cognate NF- B Binding Sites in the Enhancer (original) (raw)
Related papers
2006
The role of NF-B in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-B response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEPenhancer that, although this region is essential for HCMV growth, none of the four NF-B response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.
Journal of Virology, 2004
shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-B pathway and binding of NF-B to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.
Journal of Virology, 2004
We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-B. Here, we investigate the mechanism for the HCMV-induced NF-B activation and the role that the induced NF-B plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-B, the IB-superrepressor, we demonstrated that active NF-B is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-B activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-B, IB␣ and IB. Because the IB kinases (IKKs) regulate the degradation of the IBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-B plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-B activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.
Virus Research, 1996
The transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by a large and complex enhancer containing several consensus binding sites for the ubiquitous transcription factor NF-hB. To verify whether MCMV, like the human CMV, can activate NF-xB-dependent transcription, we transfected murine embryo fibroblasts cells with a construct containing three copies of the NF-xB element in front of the homologous minimal MCMV IE1-3 promoter. Upon MCMV infection the reporter gene activity was transactivated to about three-fold above the basal level. The specificity of this transactivation was demonstrated by the lack of any significant effect on the activity of DNA constructs containing either a mutated NF-xB trimer or an ATF/CRE trimer. Gel shift assays with a NF-KB probe revealed that MCMV infection activated DNA binding proteins showing NF-xB characteristics. The DNA-binding activity remained elevated during the course of infection and was associated to an increase in the steady-state mRNA levels for the NF-tcB subunit p105/p50. Since the promoter of the p105/p50 gene was transactivated by MCMV infection during the period in which the IE proteins are expressed, the role of the two major IE transcriptional regulatory proteins was examined. In cotransfection experiments, the IE1 protein transactivated the p105/p50 promoter, whereas the IE3 was ineffective in increasing the transcription of the reporter gene. Taken as a whole, these results demonstrate that MCMV, like its human counterpart, regulates the cellular NF-xB activity needed for the initial induction of the IE genes and the progression of the viral replicative cycle.
Journal of Virology, 2010
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-B binding sites within the MIEP enhancer and cellular NF-B 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.
Virology, 2006
Infection of fibroblasts by human cytomegalovirus (HCMV) rapidly activates the NF-κB signaling pathway, which we documented promotes efficient transactivation of the major immediate-early promoter (DeMeritt et al., 2004. J. Virol. 78: 4498-4507). Because a second, sustained increase in NF-κB activity following the initial phase of NF-κB activation was also observed, we investigated the role that this prolonged NF-κB activation played in viral replication and late gene expression. We first investigated HCMV replication in cells in which NF-κB activation was blocked by pretreatment with NF-κB inhibitors: HCMV replication was significantly decreased in these cultures. A decrease in replication was also observed when NF-κB was inhibited up to forty-eight hours post infection, suggesting a previously unidentified role for NF-κB in the regulation of the later class of viral genes.
Journal of Virology, 2002
We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements.
Journal of Virology, 2010
Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viral immediate-early (IE) genes. Accordingly, the major IE promoter (MIEP) enhancer, a critical control region for initiating lytic HCMV infection and reactivation from the latent state, contains one well-characterized AP-1 site and a second candidate interaction site. In this study we explored the role of these AP-1 elements in the context of the infection. We first show that the distal candidate AP-1 motif binds c-Fos/c-Jun heterodimers (AP-1 complex) and confers c-Fos/c-Jun-mediated activity to a core promoter. Site-directed mutagenesis studies indicate that both AP-1 response elements are critical for 12- O -tetradecanoylphorbol-13-acetate (TPA)-enhanced MIEP activity in transient-transfection assays. In marked contrast to the results obtained with the isolated promoter, disruption of the AP-1 re...