AbstracteIF3 in mammals is the largest translation initiation factor (~800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited capdependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ~40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.Eukaryotic translation initiation involves numerous initiation factors (eIFs)2 that participate in recruitment of initiator tRNA and mRNA to the 40 S ribosomal subunit, recognition of the initiator AUG codon, and joining of the 40 S and 60 S ribosomal subunits, culminating in formation of the first peptide bond (1). The factors required for recruitment of mRNA include eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF4H, and PABP. eIF4E and PABP bind the 5′ cap and 3′ poly(A) tract of mRNA, respectively, whereas eIF4A unwinds 5′-terminal secondary structure in an ATP-dependent process that also involves the RNA-binding proteins eIF4B and eIF4H. eIF4G forms specific complexes with eIF4E, eIF4A, and PABP, thereby linking the processes of cap recognition, poly(A) binding, and secondary structure melting. eIF4G in turn is recruited to the 40 S ribosomal subunit via binding to the multisubunit complex eIF3.* This work was supported by National Institutes of Health Grants GM20818 (to R. E. R.) and GM22135 (to J. W. B. H.).1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ...

The oral cavity is a persistent reservoir for Epstein-Barr virus (EBV) with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns regulated by epigenetic modifications involving DNA methylation and chromatin structure. Regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may result in long-lasting host epigenetic reprogramming. To identify epigenetic events following EBV infection, a transient infection model was established to map epigenetic changes in telomerase-immortalized oral keratinocytes. EBV-infected oral keratinocytes exhibited a predominantly latent viral gene expression program with some lytic or abortive replication. Calcium and methylcelluloseinduced differentiation was delayed in EBV-positive clones and in clones that lost EBV compared to uninfected controls, indicating a functional consequence of EBV epigenetic modifications. Analysis of global cellular DNA methylation identified over 13,000 differentially methylated CpG residues in cells exposed to EBV compared to uninfected controls, with CpG island hypermethylation observed at several cellular genes. Although the vast majority of the DNA methylation changes were silent, 65 cellular genes that acquired CpG methylation showed altered transcript levels. Genes with increased transcript levels frequently acquired DNA methylation within the gene body while those with decreased transcript levels acquired DNA methylation near the transcription start site. Treatment with the DNA methyltransferase inhibitor, decitabine, restored expression of some hypermethylated genes in EBV-infected and EBV-negative transiently infected clones. Overall, these observations suggested that EBV infection of keratinocytes leaves a lasting epigenetic imprint that can enhance the tumorigenic phenotype of infected cells. IMPORTANCEHere, we show that EBV infection of oral keratinocytes led to CpG island hypermethylation as an epigenetic scar of prior EBV infection that was retained after loss of the virus. Such EBV-induced epigenetic modification recapitulated the hypermethylated CpG island methylator phenotype (CIMP) observed in EBV-associated carcinomas. These epigenetic alterations not only impacted gene expression but also resulted in delayed calcium and methylcellulose-induced keratinocyte differentiation. Importantly, these epigenetic changes occurred in cells that were not as genetically unstable as carcinoma cells, indicating that EBV infection induced an epigenetic mutator phenotype. The impact of this work is that we have provided a mechanistic framework for how a tumor virus using the epigenetic machinery can act in a "hit-and-run" fashion, with retention of epigenetic alterations after loss of the virus. Unlike genetic alterations, these virally induced epigenetic changes can be reversed pharmacologically, providing therapeutic interventions to EBV-associated malignancies. E pstein-Barr virus (EBV) is a prevalent gammaherpesvirus i...

Background: Retinoids have been studied extensively for their potential as therapeutic and chemopreventive agents for a variety of cancers, including nonmelanoma skin cancer (NMSC). Despite their use for many years, the mechanism of action of retinoids in the prevention of NMSC is still unclear. In this study we have attempted to understand the chemopreventive mechanism of all-trans retinoic acid (ATRA), a primary biologically active retinoid, in order to more efficiently utilize retinoids in the clinic.

Background Myocarditis is an inflammatory disease of the cardiac muscle and is mainly caused by viral infections. Viral myocarditis has been proposed to be divided into 3 phases: the acute viral phase, the subacute immune phase, and the chronic cardiac remodeling phase. Although individualized therapy should be applied depending on the phase, no clinical or experimental studies have found biomarkers that distinguish between the 3 phases. Theiler’s murine encephalomyelitis virus belongs to the genus Cardiovirus and can cause myocarditis in susceptible mouse strains. Methods and Results Using this novel model for viral myocarditis induced with Theiler’s murine encephalomyelitis virus, we conducted multivariate analysis including echocardiography, serum troponin and viral RNA titration, and microarray to identify the biomarker candidates that can discriminate the 3 phases. Using C3H mice infected with Theiler’s murine encephalomyelitis virus on 4, 7, and 60 days post infection, we conducted bioinformatics analyses, including principal component analysis and k-means clustering of microarray data, because our traditional cardiac and serum assays, including 2-way comparison of microarray data, did not lead to the identification of a single biomarker. Principal component analysis separated heart samples clearly between the groups of 4, 7, and 60 days post infection. Representative genes contributing to the separation were as follows: 4 and 7 days post infection, innate immunity–related genes, such as Irf7 and Cxcl9; 7 and 60 days post infection, acquired immunity–related genes, such as Cd3g and H2-Aa; and cardiac remodeling–related genes, such as Mmp12 and Gpnmb. Conclusions Sets of molecules, not single molecules, identified by unsupervised principal component analysis, were found to be useful as phase-specific biomarkers.

Epstein-Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV’s contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV-negative, transiently infected clones to uninfected controls identified expression changes in over 1000 genes. Among downregulated genes, several genes known to be DNA methylated in cancer were identified including E-cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV-positive cells were retained following viral loss indicating an epigenetic effect. Repression of PYCARD expression was due to increased promoter CpG methylation, whereas loss of E-cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E-cadherin promoter. Rather, repression of E-cadherin was consistent with formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5’ end of the E-cadherin gene was observed in an EBV-negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, while its own contribution to the oncogenic process is masked.