Two Sp1/Sp3 Binding Sites in the Major Immediate-Early Proximal Enhancer of Human Cytomegalovirus Have a Significant Role in Viral Replication

Abstract

Human cytomegalovirus (HCMV), a member of the betaherpesvirus family, infects most individuals asymptomatically. The virus persists in CD34ϩ hematopoietic progenitor cells, monocytes, and CD34 ϩ -derived dendritic cells from healthy seropositive individuals (22,36,37). Reactivation of HCMV is related to a variety of diseases such as pneumonitis, hepatitis, and retinitis (7, 25). The mechanisms underlying maintenance of the latent viral genome and the switch from the latent to lytic forms of infection remain unclear.The host range of HCMV is narrow in cell culture. The virus replicates productively in terminally differentiated cells such as fibroblasts, epithelial and endothelial cells, and monocyte-derived macrophages (17,18,27,41,59,60,65). The major immediate-early (MIE) genes of HCMV play a key role in determining the efficiency of viral replication. IE1 (UL123) and IE2 (UL122) encode pIE72 and pIE86 proteins important for regulation of subsequent viral gene expression. The pIE72 protein contributes to efficient viral replication at low multiplicity of infection (MOI) (19,21). The IE86 protein is essential for early viral gene expression and autoregulates transcription of the IE1 and IE2 genes (12,43,47,56).The region upstream of the HCMV MIE promoter is divided into three regions: the modulator, the unique region, and the enhancer (52, 61). The modulator has no effect on MIE transcription and on viral replication in cell culture (51). The unique region also has no effect on transcription from the MIE promoter, but one or more cis-acting elements repress transcription from the divergent early viral UL127 promoter (39, 45). The enhancer is divided into distal and proximal components (50). Without the distal enhancer the recombinant virus replicates slowly and has a small-plaque phenotype in human fibroblast cells (50). Proximal and distal chimeras of the human and murine CMV enhancers replicate less efficiently at low MOIs and demonstrate the small-plaque phenotype (29). The entire enhancer region of human CMV is required for robust MIE gene expression.In a previous report, we demonstrated a direct correlation between the extent of the proximal enhancer and the amount of MIE gene transcription and the level of infectious virus replication (30). Deletion of the enhancer from nucleotide position Ϫ636 to Ϫ39 resulted in no replication in human foreskin fibroblasts (HFFs). In contrast, a recombinant virus with a deletion from position Ϫ636 to Ϫ67 replicated independently. This recombinant virus contained a single GC box upstream of the MIE promoter that binds the Sp1 transcription factor (30).The Sp1 family of transcription factors is composed of four proteins (Sp1, Sp2, Sp3, and Sp4) (64). In addition to a highly conserved DNA-binding domain, all four cellular proteins have glutamine-rich activation domains adjacent to serine/ threonine-rich stretches in their N-terminal regions. Sp1, Sp2, and Sp3 are ubiquitously expressed, but expression of Sp4 is limited to the brain (63). Sp1 and Sp3 both recognize GC-rich se...

Sp3/REST/HDAC1/HDAC2 Complex Represses and Sp1/HIF-1/p300 Complex Activates ncx1 Gene Transcription, in Brain Ischemia and in Ischemic Brain Preconditioning, by Epigenetic Mechanism

“…Sp1 and Sp3 bind to a specific region of ncx1-Br promoter sequence Because Sp1 and Sp3 both recognize the GC-rich sequences known as GC boxes (Isomura et al, 2005), we searched for Sp1 sequences at the level of the ncx1-Br promoter (GenBank accession no. U95138) and subjected the ncx1-Br sequence to a computational prediction of transcriptional factors using the database TF SEARCH, version 1.3 (Cassimere et al, 2009).…”

The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication

“…After infection at a multiplicity of infection (MOI) of 3 or 0.01, cells were collected at 6 h and 2, 3, and 4 days postinfection (dpi). Cells in 35-mm plates in triplicate were suspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, and 20 g/ml RNase A) containing 50 g/ml proteinase K. The input and replicated viral DNAs were detected by real-time PCR using HCMV gB primers and probe as described previously (20,24). Real-time PCR with 18S rRNA gene primers and probe purchased from Applied Biosystems (Foster City, CA) was also performed to serve as an internal control for input DNA.…”

“…Cells were harvested at the indicated times postinfection, washed with phosphate-buffered saline (PBS), and treated with lysis buffer as described previously (19,20). Twenty-microgram aliquots of proteins were loaded into each lane for sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes.…”

“…The major IE genes (MIE) UL123 and UL122 (IE1/IE2) play a critical role in subsequent viral gene expression and the efficiency of viral replication (19,20,24,(33)(34)(35). The early viral genes encode proteins necessary for viral DNA replication (36).…”

DNA Polymerase Processivity Factor of Human Cytomegalovirus May Be a Key Molecule for Molecular Coupling of Viral DNA Replication to Transcription

“…or 5 and 8 d.p.i.., respectively. Cells in 35 mm plates in triplicate were suspended in lysis buffer (50mM Tris-HCl, pH 8.0, 10mM EDTA, 1% SDS and 20µg/ml RNase A) containing 50 µg/ml proteinase K. The replicated viral DNA was quantitated by real-time PCR using HCMV gB primers and probes as described previously (Isomura, Tsurumi et al 2004;Isomura, Stinski et al 2005). Real-time PCR with 18S primers and probe purchased from Applied Biosystems (Foster City, CA.)…”