Biology subjects

Groves, I. J.

Publications and source records attributed to Groves, I. J..

6 recordsLinked to original sources

bioRxiv2025

Human cytomegalovirus-encoded G protein-coupled receptor (GPCR), UL78, regulates viral reactivation

Human cytomegalovirus (CMV) is a ubiquitous pathogen that establishes life-long, latent infection in hematopoietic cells. Immune-competent individuals are usually asymptomatic for disease. However, immune dysregulation in latently-infected individuals can result in viral reactivation, often causing further complications. Viral gene transcription during latency is restricted, although the CMV-encoded G-protein coupled receptor homologs, US28 and UL78, are expressed. We and others find US28 is critical for establishing and maintaining viral latency, in part, through regulating host cell signaling. How US28 switches from pro-latent to pro-lytic during reactivation, however, is unknown, though our findings herein reveal a role for UL78. Myeloid cells infected with a UL78 ORF deletion mutant maintain viral latency yet fail to efficiently reactivate. However, the UL78 G protein-coupling domain is not required for reactivation, suggesting UL78-mediated signaling is not critical for reactivation. Prior work revealed UL78 and US28 interact, resulting in altered US28-mediated signaling. Additionally, we showed US28 attenuates ERK phosphorylation during latency, while ERK is phosphorylated upon reactivation; however, the mechanism underlying this switch is unknown. Thus, we hypothesized the UL78:US28 interaction is important for altering US28-mediating signaling upon viral reactivation. We find US28 and UL78 interact during lytic infection of fibroblasts and colocalize in myeloid cells upon their differentiation. Further, reactivation in myeloid cells latently-infected with wild-type virus results in upregulated ERK phosphorylation, while parallel cultures infected with the UL78-deficient virus fail to do so. Our data reveal the first function for UL78 in myeloid cells, where it influences cellular signaling to switch from pro-latent to pro-lytic. ImportanceCytomegalovirus (CMV) is a ubiquitous human herpesvirus, infecting the majority of the population worldwide. As with all herpesviruses, once an individual is infected with CMV, the virus remains in a persons blood cells for their life in a silenced state called latency, and this infection, for the most part, remains asymptomatic. When an infected individuals immune system fails to function properly, however, CMV can become active (termed viral reactivation), which allows the virus to replicate and cause downstream disease. Our understanding of the cellular and viral factors that dictate this switch from silenced to activated remains incomplete. Here, we show a viral protein, UL78, is required for this switch. We find UL78 helps to reshape cellular signaling, changing the cell environment from one that favors latency to one that instead supports reactivation. This highlights a new avenue for therapeutic intervention to prevent CMV reactivation and downstream disease.

microbiology↗

bioRxiv2023

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. The HCMV major immediate early promoter (MIEP)/enhancer is a key factor in this process, as its transactivation from a repressed to active state helps drive viral gene transcription necessary for reactivation from latency. Numerous host transcription factors bind the MIE locus and recruit repressive chromatin modifiers, thus impeding virus reactivation. One such factor is CCCTC-binding protein (CTCF), a highly conserved host zinc finger protein that mediates chromatin conformation and nuclear architecture. However, the mechanisms by which CTCF contributes to HCMV latency were previously unexplored. Here, we confirm CTCF binds two convergent sites within the MIE locus during latency in primary CD14+ monocytes, and following cellular differentiation, CTCF association is lost as the virus reactivates. While mutation of the MIE enhancer CTCF binding site does not impact viral lytic growth in fibroblasts, this mutant virus fails to maintain latency in myeloid cells. Furthermore, we show the two convergent CTCF binding sites allow looping to occur across the MIEP, supporting transcriptional repression during latency. Indeed, looping between the two sites diminishes during virus reactivation, concurrent with activation of MIE transcription. Taken together, our data reveal that three-dimensional chromatin looping aids in the regulation of HCMV latency, and provides insight into promoter/enhancer regulation that may prove broadly applicable across biological systems. Significance StatementHuman cytomegalovirus (HCMV) remains an important healthcare consideration driven by disease in at-risk populations associated with reactivation of this virus from latent infection. We show here the establishment of latency is aided by a host nuclear architectural protein, CTCF. By binding two convergent sites on the virus major immediate early promoter/enhancer region, which largely acts as a switch from latency to reactivation, CTCF anchors a chromatin loop such that the virus promoter is maintained in a transcriptionally repressed state. Upon differentiation of cells, CTCF protein levels decrease, and this loop is alleviated as the virus reactivates. Our findings reveal further insight into the regulation of HCMV latency and promoter/enhancer elements, which is broadly applicable across biological systems.

microbiology↗

bioRxiv2021

CTCF association with episomal HPV16 genomes regulates viral oncogene transcription and splicing

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.

cancer biology↗

bioRxiv2021

Three-dimensional 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. 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) and long-range (>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.

cancer biology↗

bioRxiv2018

Increased Epstein-Barr virus C-promoter activity with CTCF-binding site deletion is associated with elevated EBNA2 recruitment.

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-RBPJ{kappa} 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 RBPJ{kappa}, 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.

microbiology↗

bioRxiv2017

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naive 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 is reported to enhance gene activation by the EBV protein EBNA2 in vitro.\n\nWe 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.\n\nQuantitative 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.\n\nWe 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.\n\nAuthor 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.\n\nWe 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.\n\nThis 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.

microbiology↗