Upon herpesvirus infection, viral DNA becomes associated with nuclear structures known as nuclear domain 10 (ND10). The role of ND10 during herpesvirus infection has long been contentious; data arguing for a role for ND10 in repression of infection have been countered by other data showing little effect of ND10 on virus infection. Here we show that knockdown of human Daxx (hDaxx) expression, an important component of ND10, prior to infection with human cytomegalovirus resulted in increased levels of viral immediate early RNA and protein expression and that this correlated with an increased association of the major immediate early promoter with markers of transcriptionally active chromatin. Conversely, we also show that stable overexpression of hDaxx renders cells refractory to cytomegalovirus immediate early gene expression. Intriguingly, this hDaxxmediated repression appears to be restricted to cells stably overexpressing hDaxx and is not recapitulated in transient transfection assays. Finally, hDaxx-mediated repression of cytomegalovirus major immediate early gene expression was overcome by infecting at higher virus titers, suggesting that an incoming viral structural protein or viral DNA is responsible for overcoming the repression of viral gene expression in hDaxx superexpressing cells. These data suggest that hDaxx in ND10 functions at the site of cytomegalovirus genome deposition to repress transcription of incoming viral genomes and that this repression is mediated by a direct and immediate effect of hDaxx on chromatin modification around the viral major immediate early promoter. Human cytomegalovirus (HCMV)3 is an extremely widespread, opportunistic pathogen of considerable clinical significance. Primary infection of healthy, immunocompetent individuals with HCMV is normally asymptomatic. However, as is the case with all herpesviruses, after the initial control of primary infection by the immune system, HCMV establishes lifelong latency in the host. During latency and throughout the lifetime of the infected individual, the herpesviruses can reactivate. Although generally asymptomatic, such reactivation can result in severe disease. As such, the disease burden of herpesviruses can be life-long and particularly severe when associated with immunosuppression, such as in transplant patients and in human immunodeficiency virus-infected patients who have developed immunodeficiency (1, 2).After infection with herpes simplex virus-1, the viral genomes appear to co-localize with cellular nuclear structures known as nuclear domain 10 (ND10) and give rise to replication centers in close proximity to these sites (3, 4). ND10 are discrete interchromosomal accumulations of a number of proteins, many of which are transcriptional repressors. Intriguingly, many of the human herpesviruses encode an immediate early protein, which functions to destroy or redistribute components of ND10 (5-17), and a number of studies have attempted to address the role of ND10 during infection. One of the first observations was that ICP0 (int...

Epstein-Barr virus (EBV) is able to drive the transformation of B-cells, resulting in the generation of lymphoblastoid cell lines (LCLs) in vitro. EBV nuclear proteins EBNA3A and EBNA3C are necessary for efficient transformation, while EBNA3B is dispensable. We describe a transcriptome analysis of BL31 cells infected with a series of EBNA3-knockout EBVs, including one deleted for all three EBNA3 genes. Using Affymetrix Exon 1.0 ST microarrays analysed with the MMBGX algorithm, we have identified over 1000 genes whose regulation by EBV requires one of the EBNA3s. Remarkably, a third of the genes identified require more than one EBNA3 for their regulation, predominantly EBNA3C co-operating with either EBNA3B, EBNA3A or both. The microarray was validated by real-time PCR, while ChIP analysis of a selection of co-operatively repressed promoters indicates a role for polycomb group complexes. Targets include genes involved in apoptosis, cell migration and B-cell differentiation, and show a highly significant but subtle alteration in genes involved in mitosis. In order to assess the relevance of the BL31 system to LCLs, we analysed the transcriptome of a set of EBNA3B knockout (3BKO) LCLs. Around a third of the genes whose expression level in LCLs was altered in the absence of EBNA3B were also altered in 3BKO-BL31 cell lines.Among these are TERT and TCL1A, implying that EBV-induced changes in the expression of these genes are not required for B-cell transformation. We also identify 26 genes that require both EBNA3A and EBNA3B for their regulation in LCLs. Together, this shows the complexity of the interaction between EBV and its host, whereby multiple EBNA3 proteins co-operate to modulate the behaviour of the host cell.

Keywords: human papillomavirus; mucosa; oncogene; E6/E7; epigenetics; integration; squamous cell carcinoma Human papillomavirus infection and global diseaseHuman papillomavirus (HPV) infection poses a significant risk to morbidity and mortality worldwide, being associated with ∼4.8% of all human cancers [1]. Papillomaviruses are a family of small, non-enveloped viruses with a double-stranded DNA (dsDNA) genome of, in the case of HPV, approximately 7.9 kilobases (kb) [2]. To date, > 180 individual types of HPV have been sequenced [3,4] and all infect epithelial cells, usually with a preference for either cutaneous or mucosal surfaces. The proportion that infects the genital tract (30-40 types from the α-genus) can be subdivided into low-and high-risk types, based on their oncogenic potential. Low-risk HPV types, including HPV6 and 11, are associated more with benign ano-genital warts or condylomata, whereas at least 12 high-risk HPV (HRHPV) types, HPV16,, are associated with ano-genital cancers and precursor neoplastic lesions [5,6].Although HPV infection is nearly ubiquitous, the virus does not cause cancer in the large majority of cases [7]. Most infections are inapparent and cleared by the host immune system within 18 months [8]. However, ∼10-15% of women do not clear HPV infection, with persistence of high-risk HPV being the major risk factor for development of ano-genital cancers [9,10].

Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.IMPORTANCE The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity.

The complex life cycle of oncogenic human papillomavirus (HPV) initiates in undifferentiated basal epithelial keratinocytes where expression of the E6 and E7 oncogenes is restricted. Upon epithelial differentiation, E6/E7 transcription is increased through unknown mechanisms to drive cellular proliferation required to support virus replication. We report that the chromatin-organising CCCTC-binding factor (CTCF) promotes the formation of a chromatin loop in the HPV genome that epigenetically represses viral enhancer activity controlling E6/E7 expression. CTCF-dependent looping is dependent on the expression of the CTCF-associated Yin Yang 1 (YY1) transcription factor and polycomb repressor complex (PRC) recruitment, resulting in trimethylation of histone H3 at lysine 27. We show that viral oncogene up-regulation during cellular differentiation results from YY1 down-regulation, disruption of viral genome looping, and a loss of epigenetic repression of viral enhancer activity. Our data therefore reveal a key role for CTCF-YY1–dependent looping in the HPV life cycle and identify a regulatory mechanism that could be disrupted in HPV carcinogenesis.