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PDF - 62K, Supplementary Table 1: Clinico-pathological data for sample set-3. Supplementary Table 2: Primers used for mRNA qRT-PCR. Supplementary Table 3: Expression of let-7 family microRNAs in pediatric malignant-GCTs. Supplementary Table 4: Let-7 mRNA targets identified in combined microarray analysis of pediatric and adult malignant-GCTs. Supplementary Table 5: The six let-7 mRNA targets selected following microarray and qRT-PCR analysis.

PDF - 59K.

PDF - 492K, Supplementary Figure 1: Levels of the nine members of the let-7 family in pediatric malignant-GCTs. Supplementary Figure 2: PCR quantification of let-7e, LIN28 and LIN28B in malignant-GCTs.Supplementary Figure 3: Sylamer analysis of up-regulated genes in pediatric and adult malignant-GCTs. Supplementary Figure 4: Correlations between levels of let-7 and mRNA targets. Supplementary Figure 5: PCR quantification of HMGA2 in pediatric malignant-GCTs. Supplementary Figure 6: PCR validation of expression of selected let-7 mRNA targets in pediatric malignant-GCTs. Supplementary Figure 7: Effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 8: Independent confirmation of specific effects of LIN28 depletion in malignant-GCT cells. Supplementary Figure 9: Effects of let-7e mimic in malignant-GCT cells. Supplementary Figure 10: Relationships between MYCN and C-MYC versus LIN28 and LIN28B in malignant-GCTs. Supplementary Figure 11: MYCN depletion in malignant-GCTs (S11). Supplementary Figure 12: Schematic of the LIN28/let-7 axis in malignant-GCTs.

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. These ex vivo data correlate with our retrospective analyses of the Explorys electronic health record database, where we find dasatinib treatment is associated with a significant risk of CMV-associated disease (odds ratio 1.58, P = 0.0004). Collectively, our findings elucidate a signaling pathway that plays a central role in the balance between CMV latency and reactivation and identifies a common therapeutic cancer treatment that elevates the risk of CMV-associated disease.

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. Furthermore, using our ‘HPV16-specific Region Capture Hi-C’ technique, we have determined that chromatin interactions between the integrated virus genome and host chromosomes, both at short- (500 kbp), appear to drive local host gene dysregulation through the disruption of host:host interactions within (but not exceeding) host structures known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a ‘cancer-causing gene’ is not essential to influence their expression and that these modifications to genome interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression., Author summary The integration of human papillomaviruses (HPVs) into host chromosomes is a major feature of HPV-associated cancers, however the process by which this occurs and subsequently drives carcinogenesis is incompletely understood. Here, we devised a state-of-the-art HPV16 genome-specific DNA capture technology to precisely determine the host integration sites at a nucleotide resolution, such that we confirm the mechanism of microhomology-mediated repair (MHMR) during both ‘direct’ and ‘looping’ integration of HPV16 genomes into the host. Furthermore, our technology detects both short- and long-range interactions between HPV16 and host chromatin after virus integration, which correlates with dysregulation of host gene expression at distances up to 500kbp from the integration site. This means that HPV16 genomes can directly affect host gene expression much further away on host chromosomes than initially thought, which may lead to competitive growth advantages for certain integrated clones. Therefore, our study provides further insight into the mechanisms by which papillomaviruses are able to initiate and drive cervical carcinogenesis at an early stage after HPV integration.

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. In addition, we define a crucial role for the transcription factor Yin Yang 1 (YY1) in HCMV latency; high levels of YY1 are maintained in latently infected cells as a result of BMPR2 signaling through the SMAD4/SMAD6 axis. Activation of SMAD4/6, through BMPR2, inhibits TGFbeta receptor signaling, which leads to the degradation of YY1 via induction of a cellular microRNA (miRNA), hsa-miR-29a. Pharmacological targeting of BMPR2 in progenitor cells results in the degradation of YY1 and an inability to maintain latency and renders cells susceptible to T cell killing. These data argue that BMPR2 plays a role in HCMV latency and is a new potential therapeutic target for maintaining or disrupting HCMV latency in myeloid progenitors. IMPORTANCE Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals. Therefore, this work identifies key host-virus interactions which regulate HCMV latent infection. It also demonstrates a potential new therapeutic approach to reduce HCMV reactivation-mediated disease by the treatment of donor stem cells/organs prior to transplantation, which could have a major impact in the transplant disease setting.

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. Furthermore, using our ‘HPV16-specific Region Capture Hi-C’ technique, we have determined that three-dimensional (3D) interactions between the integrated virus genome and host chromosomes, both at short- (500 kbp), appear to drive host gene dysregulation through the disruption of local host:host 3D interactions known as topologically associating domains (TADs). This mechanism of HPV-induced host gene expression modulation indicates that integration of virus genomes near to or within a ‘cancer-causing gene’ is not essential to influence such genes within an entire TAD and that these modifications to 3D interactions could have a major role in selection of HPV integrants at the early stage of cervical neoplastic progression.

Human papillomavirus 16 (HPV16) is a high-risk alphapapillomavirus that is associated with cancers of mucosal epithelia. The virus genome exists in cells as an episome but can integrate and overexpress the E6 and E7 viral oncogenes. In related high-risk family members HPV18 and HPV31, host proteins including CTCF, an insulator, and SMC1A, a component of the cohesion complex, are known to interact with the viral genome and alter transcriptional activity, splicing patterns and episome amplification. However, the roles of these two proteins during HPV16 infection has not yet been fully examined. Here, we show during differentiation of the episomal HPV16-containing W12 cell line that CTCF association increases with the virus genome at the known E2 binding site, whilst additional CTCF binding now occurs at the putative L2 binding site, with SMC1A association occurring unchanged here. While expression of virus late transcripts (E4^L1, L2, L1) is stimulated, early transcript levels decrease by 48 hours, with the exception of the E6*IV spliced transcript. Conversely, in undifferentiated, monolayer W12 cells, CTCF knockdown increases the level of all early transcripts, whereas E6*IV level increases. Additionally, CTCF ablation as well as SMC1A knockdown results in decreases to HPV16 genome copy number. Taken together, this supports the model that while CTCF and SMC1A have a role in HPV16 genome maintenance, CTCF plays a greater part in regulating HPV16 oncogene splicing and expression during the natural lifecycle of the virus, and may be involved in a reduced risk of cancer development during episomal HPV16 infections.

Significance Human cytomegalovirus (HCMV) reactivation is a major cause of posttransplant morbidity/mortality. One approach toward reducing this is purging the transplant donor and/or recipient of latently infected cells prior to stem-cell or organ harvest/engraftment. Our findings show the involvement of host bromodomain (BRD) proteins in regulation of HCMV latency and reactivation. Bromodomain and extra-terminal inhibitor (I-BET) treatment of latently infected cells causes reactivation of lytic gene expression by release of transcription activator P-TEFb (CDK9/CycT1) from BRD4-associated repressive complexes, with subsequent recruitment via host superelongation complex (SEC). This results in immune targeting and T cell-mediated killing of these otherwise latently infected cells and provides a therapeutic “shock and kill” strategy that could reduce HCMV-mediated disease in the transplant setting., 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. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, give rise to nonpermissive infections. This aspect has been used experimentally to model latent infection, which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.

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. We have analyzed the effect of HCMV latent infection on the secretome of CD14+ monocytes, identifying an upregulation of both CCL8 and CXCL10 chemokines in the CD14+ latency-associated secretome. Unlike CD34+ cells, the CD14+ latency-associated secretome did not induce migration of resting immune cell subsets but did induce migration of activated NK and T cells expressing CXCR3 in a CXCL10 dependent manner. As reported in CD34+ latent infection, the CD14+ latency-associated secretome also suppressed the anti-viral activity of stimulated CD4+ T cells. Surprisingly, however, co-culture of activated autologous CD4+ T cells with latently infected monocytes resulted in reactivation of HCMV at levels comparable to those observed using M-CSF and IL-1β cytokines. We propose that these events represent a potential strategy to enable HCMV reactivation and local dissemination of the virus at peripheral tissue sites. post-print 2637 KB

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. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we demonstrate that (primary) cytotoxic lymphocytes can block HCMV dissemination independent of host cell death, and interferon-α/β/γ. Prior to killing, cytotoxic lymphocytes induce the degradation of viral immediate-early (IE) proteins IE1 and IE2 in HCMV-infected cells. Intriguingly, both IE1 and/or IE2 are directly proteolyzed by all human granzymes, with GrB and GrM being most efficient. GrB and GrM cleave IE1 after Asp398 and Leu414, respectively, likely resulting in IE1 aberrant cellular localization, IE1 instability, and functional impairment of IE1 to interfere with the JAK-STAT signaling pathway. Furthermore, GrB and GrM cleave IE2 after Asp184 and Leu173, respectively, resulting in IE2 aberrant cellular localization and functional abolishment of IE2 to transactivate the HCMV UL112 early promoter. Taken together, our data indicate that cytotoxic lymphocytes can also employ noncytotoxic ways to control HCMV infection, which may be explained by granzyme-mediated targeting of indispensable viral proteins during lytic infection., Author summary Human cytomegalovirus (HCMV) is the leading viral cause of congenital defects, can trigger disease in immune-compromised patients, and plays roles in cancer development. Cytotoxic lymphocytes kill HCMV-infected cells via releasing a set of five cytotoxic serine proteases called granzymes. However, the killing capacity of cytotoxic cells is limited and multiple T cell hits are required to kill a single virus-infected cell. This raises the question whether cytotoxic lymphocytes can use granzymes to control HCMV infection in a noncytotoxic manner. Here, we show that cytotoxic lymphocytes can also use granzymes to inhibit HCMV replication in absence of cell death. All five granzymes cleave and inactivate both viral immediate-early (IE1/2) proteins, which are essential players for initiating HCMV infection. Our data support the model that cytotoxic cells employ granzymes to dampen HCMV replication prior to accumulation of sufficient hits to kill the infected cell.

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. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

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. However, the mechanisms by which HRHPV integration occur and by which the subsequent gene expression changes take place are incompletely understood. The advent of next-generation sequencing (NGS) of both RNA and DNA has allowed powerful interrogation of the association of HRHPVs with human disease, including precise determination of the sites of integration and the genomic rearrangements at integration loci. In turn, these data have indicated that integration occurs through two main mechanisms: looping integration and direct insertion. Improved understanding of integration sites is allowing further investigation of the factors that provide a competitive advantage to some integrants during disease progression. Furthermore, advanced approaches to the generation of genome-wide samples have given novel insights into the three-dimensional interactions within the nucleus, which could act as another layer of epigenetic control of both virus and host transcription. It is hoped that further advances in NGS techniques and analysis will not only allow the examination of further unanswered questions regarding HPV infection, but also direct new approaches to treating HPV-associated human disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The regulation of transcription from Epstein-Barr virus promoters is known to involve the association of the host CCCTC-binding factor (CTCF) protein. This control involves direct binding of CTCF across the EBV genome and the formation of three-dimensional loops between virus promoters and enhancers. We sought to address how the deletion of a CTCF binding site upstream of the C-promoter (Cp) affected viral transcription in infected lymphoblastoid cell lines (LCLs) and how binding of the EBV trans-activating protein EBNA2 was changed across this promoter. Transcript level from Cp was up-regulated with CTCF binding site deletion, and transcription from other promoters (Wp and Qp) was decreased, while transcript levels were largely unchanged by independent mutation of a Cp-RBPJκ binding site. In turn, expression of EBNA2 protein was also increased, likely driven by increases in polycistronic EBNA2-encoding transcripts. Finally, Cp up-regulation was associated with an 8-fold increase in EBNA2 enrichment across Cp, concomitant with increased association of the associated cellular factor RBPJκ, probably due to a more accessible three-dimensional chromatin conformation upstream of Cp. Overall, the data presented here confirm that binding of CTCF directly upstream of Cp is important for the regulation of transcription from this and other EBV promoters.

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). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.

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 is reported to enhance gene activation by the EBV protein EBNA2 in vitro.We generated two sets of EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of IR1. Intronic mutations in the first of these knockouts suggested a role for the EBV sisRNAs in transformation. LPKOs with intact introns established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but umbilical cord B cells, and naive (IgD+, CD27-) adult B cells consistently died approximately two weeks after infection with LPKO, failing to establish LCLs.Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes, particularly in the first 1-2 weeks. By 30 days post infection, these levels had equalised. In contrast, EBNA2-regulated host genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that recruitment of EBNA2 and the host factors EBF1 and RBPJ to all latency promoters tested was severely delayed, whereas these same factors were recruited efficiently to several host genes, some of which exhibited increased EBNA2 recruitment.We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that different properties of EBV may have differing importance in transforming different B cell subsets.Author summaryEpstein-Barr virus (EBV) infects almost everyone. Once infected, people harbor the virus for life, shedding it in saliva. Infection of children is asymptomatic, but a first infection during adolescence or adulthood can cause glandular fever (mono). EBV is also implicated in several different cancers. EBV infection of B cells (the immune cell that produces antibodies) can drive them to replicate almost indefinitely (‘transformation’), generating cell lines. We have investigated the role of a virus protein – EBNA-LP – which is thought to support gene activation by the essential virus protein EBNA2.We have made an EBV in which the EBNA-LP gene has been disrupted. This virus (LPKO) shows several properties. 1. It is reduced in its ability to transform adult cells, while immature B cells (more frequent in the young) die two weeks after LPKO infection. 2. Some virus genes fail to turn on immediately after LPKO infection. 3. Binding of EBNA2 to these genes is delayed, as is binding of some cellular factors. 4. EBNA-LP does not affect EBNA2-targeted cellular genes in the same way.This shows that EBNA-LP is more important in immature cells, and that it regulates virus genes – but not host genes – more widely than simply through EBNA2.

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. However, the mechanisms by which HRHPV integration occur and by which the subsequent gene expression changes take place are incompletely understood. The advent of next-generation sequencing (NGS) of both RNA and DNA has allowed powerful interrogation of the association of HRHPVs with human disease, including precise determination of the sites of integration and the genomic rearrangements at integration loci. In turn, these data have indicated that integration occurs through two main mechanisms: looping integration and direct insertion. Improved understanding of integration sites is allowing further investigation of the factors that provide a competitive advantage to some integrants during disease progression. Furthermore, advanced approaches to the generation of genome-wide samples have given novel insights into the three-dimensional interactions within the nucleus, which could act as another layer of epigenetic control of both virus and host transcription. It is hoped that further advances in NGS techniques and analysis will not only allow the examination of further unanswered questions regarding HPV infection, but also direct new approaches to treating HPV-associated human disease. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

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. These early events in cervical carcinogenesis cannot readily be addressed by cross-sectional studies of clinical samples. We used the W12 model system to generate a panel of cervical squamous cell clones that were derived from an identical background under non-competitive conditions and differed only by the genomic site of HPV16 integration. Compared with the ‘baseline’ episome-containing cells from which they were isolated, only 9/17 clones (53%) showed significantly greater growth rates and only 7/17 (41%) showed significantly greater expression of the major virus oncogenes E7/E6. There were significant variations in levels of HPV16 transcription per DNA template, changes that were associated with histone modifications in the integrated virus chromatin. Cell growth rates showed only weak and non-significant associations with protein and mRNA levels for E7, E6, and the mean E7/E6 values. We conclude that HPV16 integration in basal cervical cells does not necessarily lead to increased levels of virus oncogenes, or to a competitive growth advantage, when compared with the initiating episome-containing cells. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells., Author summary Epstein-Barr virus (EBV) infects almost everyone. Once infected, people harbor the virus for life, shedding it in saliva. Infection of children is asymptomatic, but a first infection during adolescence or adulthood can cause glandular fever (infectious mononucleosis). EBV is also implicated in several different cancers. EBV infection of B cells (antibody-producing immune cells) can drive them to replicate almost indefinitely (‘transformation’), generating cell lines. We have investigated the role of an EBV protein (EBNA-LP) which is thought to support gene activation by the essential virus protein EBNA2. We have made an EBV in which the EBNA-LP gene has been disrupted. This virus (LPKO) shows several properties. 1. It is reduced in its ability to transform B cells; 2. ‘Naïve’ B cells (those whose antibodies have not adapted to fight infections) die two weeks after LPKO infection; 3. Some virus genes fail to turn on immediately after LPKO infection. 4. Binding of EBNA2 and various cellular factors to these genes is delayed. 5. EBNA-LP does not affect EBNA2-targeted cellular genes in the same way. This shows that EBNA-LP is more important in naïve B cells, and that it helps to turn on virus genes, but not cell genes.

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. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander 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. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins.

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.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at ‘pre-IE’ times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

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. Integration of virus DNA into host chromosomes is observed in the majority of cervical squamous cell carcinomas (SCCs), although in ∼15% of cases the virus remains extrachromosomal (episomal). Interestingly, not all integration events provide a growth advantage to basal cervical epithelial cells or lead to increased levels of the virus oncogenes E6 and E7, when compared with episome-containing basal cells. The factors that provide a competitive advantage to some integrants, but not others, are complex and include virus and host contributions. Gene expression from integrated and episomal HRHPV is regulated through host epigenetic mechanisms affecting the virus long control region (LCR), which appear to be of functional importance. New approaches to treating HRHPV-associated mucosal neoplasia include knockout of integrated HRHPV DNA, depletion of virus transcripts and inhibition of virus early gene transcription through targeting or use of epigenetic modifiers. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John WileySons, Ltd.

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. Indeed, a strong negative correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, as the sequence complementary to the 2 to 7 nt common let-7 seed “GAGGUA” was enriched in the 3′ untranslated regions of mRNAs upregulated in pediatric and adult malignant GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were upregulated in malignant GCT cells, confirming significant negative correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by quantitative reverse transcription PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67, and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and downregulate MYCN, AURKB, and LIN28, the latter via a double-negative feedback loop. We conclude that the LIN28/let-7 pathway has a critical pathobiologic role in malignant GCTs and therefore offers a promising target for therapeutic intervention. Cancer Res; 73(15); 4872–84. ©2013 AACR.

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. We found that while cervical neoplastic progression in vitro was characterized by dynamic changes in HPV16 transcript levels, viral early gene expression was required for cell survival at all stages of carcinogenesis, regardless of viral physical state, levels of early gene expression or histology in organotypic tissue culture. Moreover, HPV16 early gene depletion induced changes in host gene expression that were common to both episome-containing and integrant-containing cells. In particular, we observed up-regulation of autophagy genes, associated with enrichment of senescence and innate immune-response pathways, including the senescence-associated secretory phenotype (SASP). In keeping with these observations, HPV16 early gene depletion induced autophagy in both episome-containing and integrant-containing W12 cells, as evidenced by the appearance of autophagosomes, punctate expression of the autophagy marker LC3, conversion of LC3B-I to LC3B-II, and reduced levels of the autophagy substrate p62. Consistent with the reported association between autophagy and senescence pathways, HPV16 early gene depletion induced expression of the senescence marker beta-galactosidase and increased secretion of the SASP-related protein IGFBP3. Together, these data indicate that depleting HR-HPV early genes would be of potential therapeutic benefit in all cervical carcinogenesis pathways, regardless of viral physical state. In addition, the senescence/SASP response associated with autophagy induction may promote beneficial immune effects in bystander cells.

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.

Human cytomegalovirus (HCMV) regulates NF-κB during infection by a variety of mechanisms. For example, the HCMV gene product, UL144, is known to activate NF-κB in a tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-dependent manner, causing the upregulation of the chemokine CCL22 (MDC). Viral UL144 is expressed from the UL/b′ region of the HCMV genome at early times postinfection and is a TNFR1-like homologue. Despite this homology to the TNFR1 receptor superfamily, UL144 does not bind to members of the TNF ligand superfamily. We show here that the upregulation of NF-κB by UL144 is dependent upon cellular tripartite motif 23 (TRIM23) protein. We propose a mechanism by which UL144 activates NF-κB through a direct interaction with the cellular protein TRIM23 in a complex containing TRAF6. In contrast, TRIM23 is not involved in conventional double-stranded RNA signaling via NF-κB. Therefore, we present a novel role for TRIM23 that is specific to UL144-mediated activation of NF-κB during the course of virus infection.

The product of the human cytomegalovirus (HCMV) gene UL144, expressed at early times postinfection, is located in the UL/b′ region of the viral genome and is related to members of the tumor necrosis factor receptor superfamily, but it does not bind tumor necrosis factor superfamily ligands. However, UL144 does activate NF-κB, resulting in NF-κB-mediated activation of the cellular chemokine CCL22. Consistent with this finding, isolates of HCMV lacking the UL/b′ region show no such activation of CCL22. Recently, it has been suggested that activation of NF-κB is repressed by the product of the viral gene IE86: IE86 appears to block NF-κB binding to DNA but not nuclear translocation of NF-κB. Intriguingly, IE86 is detectable throughout an infection with the virus, so how UL144 is able to activate NF-κB in the presence of continued IE86 expression is unclear. Here we show that although IE86 does repress the UL144-mediated activation of a synthetic NF-κB promoter, it is unable to block UL144-mediated activation of the CCL22 promoter, and this lack of responsiveness to IE86 appears to be regulated by binding of the CREB transcription factor.

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. Findings We treated HPV16-positive cervical keratinocytes with siRNAs targeting the HPV16 E7 gene and used quantitative PCR to compare transcript levels of E7 with those of E6 and E2, viral genes located upstream and downstream of the target site respectively. We compared our findings from cDNA generated using oligo-dT primers alone with those from cDNA generated using a combination of random hexamer and oligo-dT primers. Our data show that when polycistronic transcripts are targeted by siRNAs, there is a period when untranslatable cleaved mRNA upstream of the siRNA binding site remains detectable by PCR, if cDNA is generated using random hexamer primers. Such false indications of mRNA abundance are avoided using oligo-dT primers. The period corresponds to the time taken for siRNA activity and degradation of the cleaved transcripts. Genes downstream of the siRNA binding site are detectable during this interval, regardless of how the cDNA is generated. Conclusions These data emphasise the importance of the cDNA synthesis method used when measuring transcript abundance following siRNA depletion of polycistronic transcripts. They provide a partial explanation for erroneous reports suggesting that siRNAs targeting HPV E7 can have gene-specific effects.