Loopy virus or controlled contortionist? 3D regulation of HCMV gene expression by CTCF-driven chromatin interactions.

Three-dimensional chromatin control of eukaryotic transcription is pivotal for regulating gene expression. This additional layer of epigenetic regulation is also utilized by DNA viruses, including herpesviruses. Dynamic, spatial genomic organization often involves looping of chromatin anchored by host-encoded CCCTC-binding factor (CTCF) and other factors, which control crosstalk between promoters and enhancers.

Host-encoded CTCF regulates human cytomegalovirus latency via chromatin looping.

Human cytomegalovirus (HCMV) is a prevalent pathogen that establishes life-long latent infection in hematopoietic cells. While this infection is usually asymptomatic, immune dysregulation leads to viral reactivation, which can cause significant morbidity and mortality. However, the mechanisms underpinning reactivation remain incompletely understood.

Chromatin control of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a betaherpesvirus that establishes lifelong infection in its host and can cause severe comorbidities in individuals with suppressed or compromised immune systems. The lifecycle of HCMV consists of lytic and latent phases, largely dependent upon the cell type infected and whether transcription from the major immediate early locus can ensue. Control of this locus, which acts as a critical "switch" region from where the lytic gene expression cascade originates, as well as regulation of the additional ~235 kilobases of virus genome, occurs through chromatinization with cellular histone proteins after infection.

Cytomegalovirus US28 regulates cellular EphA2 to maintain viral latency.

Cytomegalovirus (CMV) reactivation from latency following immune dysregulation remains a serious risk for patients, often causing substantial morbidity and mortality. Here, we demonstrate the CMV-encoded G protein-coupled receptor, US28, in coordination with cellular Ephrin receptor A2, attenuates mitogen-activated protein kinase signaling, thereby limiting viral replication in latently infected primary monocytes. Furthermore, treatment of latently infected primary monocytes with dasatinib, a Food and Drug Association-approved kinase inhibitor used to treat a subset of leukemias, results in CMV reactivation.

Short- and long-range cis interactions between integrated HPV genomes and cellular chromatin dysregulate host gene expression in early cervical carcinogenesis.

Development of cervical cancer is directly associated with integration of human papillomavirus (HPV) genomes into host chromosomes and subsequent modulation of HPV oncogene expression, which correlates with multi-layered epigenetic changes at the integrated HPV genomes. However, the process of integration itself and dysregulation of host gene expression at sites of integration in our model of HPV16 integrant clone natural selection has remained enigmatic. We now show, using a state-of-the-art 'HPV integrated site capture' (HISC) technique, that integration likely occurs through microhomology-mediated repair (MHMR) mechanisms via either a direct process, resulting in host sequence deletion (in our case, partially homozygously) or via a 'looping' mechanism by which flanking host regions become amplified.

Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28.

A BMPR2/YY1 Signaling Axis Is Required for Human Cytomegalovirus Latency in Undifferentiated Myeloid Cells.

Human cytomegalovirus (HCMV) presents a major health burden in the immunocompromised and in stem cell transplant medicine. A lack of understanding about the mechanisms of HCMV latency in undifferentiated CD34 stem cells, and how latency is broken for the virus to enter the lytic phase of its infective cycle, has hampered the development of essential therapeutics. Using a human induced pluripotent stem cell (iPSC) model of HCMV latency and patient-derived myeloid cell progenitors, we demonstrate that bone morphogenetic protein receptor type 2 (BMPR2) is necessary for HCMV latency.

Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation.

Human cytomegalovirus (HCMV) infection is not cleared by the initial immune response but persists for the lifetime of the host, in part due to its ability to establish a latent infection in cells of the myeloid lineage. HCMV has been shown to manipulate the secretion of cellular proteins during both lytic and latent infection; with changes caused by latent infection mainly investigated in CD34+ progenitor cells. Whilst CD34+ cells are generally bone marrow resident, their derivative CD14+ monocytes migrate to the periphery where they briefly circulate until extravasation into tissue sites.

Bromodomain proteins regulate human cytomegalovirus latency and reactivation allowing epigenetic therapeutic intervention.

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression.

Bromodomain Inhibitors as Therapeutics for Herpesvirus-Related Disease: All BETs Are Off?

Although the ubiquitous human herpesviruses (HHVs) are rarely associated with serious disease of the healthy host, primary infection and reactivation in immunocompromised individuals can lead to significant morbidity and, in some cases, mortality. Effective drugs are available for clinical treatment, however resistance is on the rise such that new anti-viral targets, as well as novel clinical treatment strategies, are required. A promising area of development and pre-clinical research is that of inhibitors of epigenetic modifying proteins that control both cellular functions and the viral life cycle.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) - the promoter that regulates immediate early (IE) gene expression - is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP.

Killer cell proteases can target viral immediate-early proteins to control human cytomegalovirus infection in a noncytotoxic manner.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and can trigger devastating disease in immune-suppressed patients. Cytotoxic lymphocytes (CD8+ T cells and NK cells) control HCMV infection by releasing interferon-γ and five granzymes (GrA, GrB, GrH, GrK, GrM), which are believed to kill infected host cells through cleavage of intracellular death substrates. However, it has recently been demonstrated that the in vivo killing capacity of cytotoxic T cells is limited and multiple T cell hits are required to kill a single virus-infected cell.

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.

Disruption of CTCF-YY1-dependent looping of the human papillomavirus genome activates differentiation-induced viral oncogene transcription.

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.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1).

Human papillomavirus genome integration in squamous carcinogenesis: what have next-generation sequencing studies taught us?

Human papillomavirus (HPV) infection is associated with ∼5% of all human cancers, including a range of squamous cell carcinomas. Persistent infection by high-risk HPVs (HRHPVs) is associated with the integration of virus genomes (which are usually stably maintained as extrachromosomal episomes) into host chromosomes. Although HRHPV integration rates differ across human sites of infection, this process appears to be an important event in HPV-associated neoplastic progression, leading to deregulation of virus oncogene expression, host gene expression modulation, and further genomic instability.

Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells.

Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available.

Pathogenesis of human papillomavirus-associated mucosal disease.

Human papillomaviruses (HPVs) are a necessary cause of carcinoma of the cervix and other mucosal epithelia. Key events in high-risk HPV (HRHPV)-associated neoplastic progression include persistent infection, deregulated expression of virus early genes in basal epithelial cells and genomic instability causing secondary host genomic imbalances. There are multiple mechanisms by which deregulated virus early gene expression may be achieved.

Virus transcript levels and cell growth rates after naturally occurring HPV16 integration events in basal cervical keratinocytes.

Cervical carcinogenesis is characterized by a clonal selection process in which the high-risk human papillomavirus (HRHPV) genome usually changes from the extra-chromosomal (episomal) state seen in productive infections to DNA that is integrated into host chromosomes. However, it is not clear whether all HRHPV integration events provide cells with a selective growth advantage compared with the episome-containing cells from which they originate. It is also unclear whether selection of cells containing a particular integrant from a mixed population simply reflects the highest levels of virus oncogene expression or has additional determinants.

Depletion of HPV16 early genes induces autophagy and senescence in a cervical carcinogenesis model, regardless of viral physical state.

In cervical carcinomas, high-risk human papillomavirus (HR-HPV) may be integrated into host chromosomes or remain extra-chromosomal (episomal). We used the W12 cervical keratinocyte model to investigate the effects of HPV16 early gene depletion on in vitro cervical carcinogenesis pathways, particularly effects shared by cells with episomal versus integrated HPV16 DNA. Importantly, we were able to study the specific cellular consequences of viral gene depletion by using short interfering RNAs known not to cause phenotypic or transcriptional off-target effects in keratinocytes.

LIN28 Expression in malignant germ cell tumors downregulates let-7 and increases oncogene levels.

Despite their clinicopathologic heterogeneity, malignant germ cell tumors (GCT) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of downregulation of the let-7 family of tumor suppressor microRNAs in malignant GCTs. Microarray results from pediatric and adult samples (n = 45) showed that LIN28, the negative regulator of let-7 biogenesis, was abundant in malignant GCTs, regardless of patient age, tumor site, or histologic subtype.

Depletion of polycistronic transcripts using short interfering RNAs: cDNA synthesis method affects levels of non-targeted genes determined by quantitative PCR.

Background: Short interfering RNAs (siRNAs) are often used to deplete viral polycistronic transcripts, such as those encoded by human papillomavirus (HPV). There are conflicting data in the literature concerning how siRNAs targeting one HPV gene can affect levels of other genes in the polycistronic transcripts. We hypothesised that the conflict might be partly explained by the method of cDNA synthesis used prior to transcript quantification.

Extensive co-operation between the Epstein-Barr virus EBNA3 proteins in the manipulation of host gene expression and epigenetic chromatin modification.

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.

Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic 'pre-immediate-early' repression of viral gene expression mediated by histone post-translational modification.

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.

Knockdown of hDaxx in normally non-permissive undifferentiated cells does not permit human cytomegalovirus immediate-early gene expression.

The cellular protein human Daxx (hDaxx), a component of nuclear domain 10 structures, is known to mediate transcriptional repression of human cytomegalovirus immediate-early (IE) gene expression upon infection of permissive cell types, at least in part, by regulation of chromatin structure around the major IE promoter (MIEP). As it is now clear that differentiation-dependent regulation of the MIEP also plays a pivotal role in the control of latency and reactivation, we asked whether hDaxx-mediated repression is involved in differentiation-dependent MIEP regulation. We show that downregulation of hDaxx by using small interfering RNA technology in undifferentiated NT2D1 cells does not permit expression of viral IE genes, nor does it result in changes in chromatin structure around the MIEP.