The human cytomegalovirus UL55 (gB) and UL75 (gH) glycoprotein ligands initiate the rapid activation of Sp1 and NF-kappaB during infection.

The cellular transcription factors Sp1 and NF-kappaB were upregulated shortly after the binding of purified live or UV-inactivated human cytomegalovirus (HCMV) to the cell surface. The rapid time frame of transcription factor induction is similar to that seen in other systems in which cellular factors are induced following receptor-ligand engagement. This similarity suggested that a cellular receptor-viral ligand interaction might be involved in Sp1 and NF-kappaB activation during the earliest stages of HCMV infection. To focus on the possible role viral ligands play in initiating cellular events following infection, we first used purified viral membrane extracts to demonstrate that constituents on the membrane are responsible for cellular activation. Additionally, these studies showed, through the use of neutralizing antibodies, that the viral membrane mediators of this activation are the major envelope glycoproteins gB (UL55) and gH (UL75). To confirm these results, neutralizing anti-gB and -gH antibodies were used to block the interactions of these glycoproteins on whole purified virus with their cell surface receptors. In so doing, we found that Sp1 and NF-kappaB induction was inhibited. Lastly, through the use of purified viral gB protein and an anti-idiotypic antibody that mimics the image of the viral gH protein, it was found that the engagement of individual viral ligands with their appropriate cell surface receptors was sufficient to activate cellular Sp1 and NF-kappaB. These results support our hypothesis that HCMV glycoproteins mediate an initial signal transduction pathway which leads to the upregulation of host cell transcription factors and suggests a model wherein the orderly sequence of virus-mediated changes in cellular activation initiates with viral binding via envelope glycoproteins to the cognate cellular receptor(s)

Induction of the transcription factor Sp1 during human cytomegalovirus infection mediates upregulation of the p65 and p105/p50 NF-kappaB promoters.

During human cytomegalovirus (HCMV) infection, the promoters for the classical NF-kappaB subunits (p65 and p105/p50) are transactivated. Previously, we demonstrated that the viral immediate-early (IE) proteins (IE1-72, IE2-55, and IE2-86) were involved in this upregulation. These viral factors alone, however, could not account for the entirety of the increased levels of transcription. Because one of the hallmarks of HCMV infection is the induction of cellular transcription factors, we hypothesized that one or more of these induced factors was also critical to the regulation of NF-kappaB during infection. Sp1 was one such factor that might be involved because p65 promoter activity was upregulated by Sp1 and both of the NF-kappaB subunit promoters are GC rich and contain Sp1 binding sites. Therefore, to detail the role that Sp1 plays in the regulation of NF-kappaB during infection, we initially examined Sp1 levels for changes during infection. HCMV infection resulted in increased Sp1 mRNA expression, protein levels, and DNA binding activity. Because both promoters were transactivated by Sp1, we reasoned that the upregulation of Sp1 played a role in p65 and p105/p50 promoter activity during infection. To address the specific role of Sp1 in p65 and p105/p50 promoter transactivation by HCMV, we mutated both promoters. These results demonstrated that the Sp1-specific DNA binding sites were involved in the virus-mediated transactivation. Last, to further dissect the role of HCMV in the Sp1-mediated induction of NF-kappaB, we examined the role that the viral IE genes played in Sp1 regulation. The IE gene products (IE1-72, IE2-55, and IE2-86) cooperated with Sp1 to increase promoter transactivation and physically interacted with Sp1. In addition, the IE2-86 product increased Sp1 DNA binding by possibly freeing up inactive Sp1. These data supported our hypothesis that Sp1 was involved in the upregulation of NF-kappaB during HCMV infection through the Sp1 binding sites in the p65 and p105/p50 promoters and additionally demonstrated a potential viral mechanism that might be responsible for the upregulation of Sp1 activity

OR14I1 is a receptor for the human cytomegalovirus pentameric complex and defines viral epithelial cell tropism

A human cytomegalovirus (HCMV) pentameric glycoprotein complex (PC), gH-gL-UL128-UL130-UL131A, is necessary for viral infection of clinically relevant cell types, including epithelial cells, which are important for interhost transmission and disease. We performed genome-wide CRISPR/Cas9 screens of different cell types in parallel to identify host genes specifically required for HCMV infection of epithelial cells. This effort identified a multipass membrane protein, OR14I1, as a receptor for HCMV infection. This olfactory receptor family member is required for HCMV attachment, entry, and infection of epithelial cells and is dependent on the presence of viral PC. OR14I1 is required for AKT activation and mediates endocytosis entry of HCMV. We further found that HCMV infection of epithelial cells is blocked by a synthetic OR14I1 peptide and inhibitors of adenylate cyclase and protein kinase A (PKA) signaling. Identification of OR14I1 as a PC-dependent HCMV host receptor associated with epithelial tropism and the role of the adenylate cyclase/PKA/AKT-mediated signaling pathway in HCMV infection reveal previously unappreciated targets for the development of vaccines and antiviral therapies

Characterization of an immediate-early gene induced in adherent monocytes that encodes IκB-like activity

We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (MAD-3) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the cdc10/ankyrin motif, which is 60% similar (46% identical) to the ankyrin repeat region of the precursor of NF-κBKBF1 p50. The C-terminus has a putative protein kinase C phosphorylation site. In vitro translated MAD-3 protein was found to specifically inhibit the DNA-binding activity of the p50p65 NF-κB complex but not that of the p50p50 KBF1 factor or of other DNA-binding proteins. The MAD-3 cDNA encodes an IκB-like protein that is likely to be involved in regulation of transcriptional responses to NF-κB, including adhesion-dependent pathways of monocyte activation

Role of human cytomegalovirus immediate-early proteins in cell growth control

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G2/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific beta-herpesvirus that, like other herpesviruses, ca

Integrins as Herpesvirus Receptors and Mediators of the Host Signalosome

The repertoire of herpesvirus receptors consists of nonintegrin and integrin molecules. Integrins interact with the conserved glycoproteins gH/gL or gB. This interaction is a conserved biology across the Herpesviridae family, likely directed to promote virus entry and endocytosis. Herpesviruses exploit this interaction to execute a range of critical functions that include (a) relocation of nonintegrin receptors (e.g., herpes simplex virus nectin1 and Kaposi's sarcoma-associated herpesvirus EphA2), or association with nonintegrin receptors (i.e., human cytomegalovirus EGFR), to dictate species-specific entry pathways; (b) activation of multiple signaling pathways (e.g., Ca2+ release, c-Src, FAK, MAPK, and PI3K); and (c) association with Rho GTPases, tyrosine kinase receptors, Toll-like receptors, which result in cytoskeletal remodeling, differential cell type targeting, and innate responses. In turn, integrins can be modulated by viral proteins (e.g., Epstein-Barr virus LMPs) to favor spread of transformed cells. We propose that herpesviruses evolved a multipartite entry system to allow interaction with multiple receptors, including integrins, required for their sophisticated life cycle