Merkel cell carcinoma (MCC) is a highly aggressive nonmelanoma skin cancer arising from epidermal mechanoreceptor Merkel cells. In 2008, a novel human polyomavirus, Merkel cell polyomavirus (MCPyV), was identified and is strongly implicated in MCC pathogenesis. Currently, little is known regarding the virus-host cell interactions which support virus replication and virus-induced mechanisms in cellular transformation and metastasis. Here we identify a new function of MCPyV small T antigen (ST) as an inhibitor of NF-κB-mediated transcription. This effect is due to an interaction between MCPyV ST and the NF-κB essential modulator (NEMO) adaptor protein. MCPyV ST expression inhibits IκB kinase α (IKKα)/IKKβ-mediated IκB phosphorylation, which limits translocation of the NF-κB heterodimer to the nucleus. Regulation of this process involves a previously undescribed interaction between MCPyV ST and the cellular phosphatase subunits, protein phosphatase 4C (PP4C) and/or protein phosphatase 2A (PP2A) Aβ, but not PP2A Aα. Together, these results highlight a novel function of MCPyV ST to subvert the innate immune response, allowing establishment of early or persistent infection within the host cell.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with multiple AIDS-related malignancies. Like other herpesviruses, KSHV has a biphasic life cycle and both the lytic and latent phases are required for tumorigenesis. Evidence suggests that KSHV lytic replication can cause genome instability in KSHV-infected cells, although no mechanism has thus far been described. A surprising link has recently been suggested between mRNA export, genome instability and cancer development. Notably, aberrations in the cellular transcription and export complex (hTREX) proteins have been identified in high-grade tumours and these defects contribute to genome instability. We have previously shown that the lytically expressed KSHV ORF57 protein interacts with the complete hTREX complex; therefore, we investigated the possible intriguing link between ORF57, hTREX and KSHV-induced genome instability. Herein, we show that lytically active KSHV infected cells induce a DNA damage response and, importantly, we demonstrate directly that this is due to DNA strand breaks. Furthermore, we show that sequestration of the hTREX complex by the KSHV ORF57 protein leads to this double strand break response and significant DNA damage. Moreover, we describe a novel mechanism showing that the genetic instability observed is a consequence of R-loop formation. Importantly, the link between hTREX sequestration and DNA damage may be a common feature in herpesvirus infection, as a similar phenotype was observed with the herpes simplex virus 1 (HSV-1) ICP27 protein. Our data provide a model of R-loop induced DNA damage in KSHV infected cells and describes a novel system for studying genome instability caused by aberrant hTREX. The hallmarks of cancer comprise the essential elements that permit the formation and development of human tumours. Genome instability is an enabling characteristic that allows the progression of tumorigenesis through genetic mutation and therefore, understanding the molecular causes of genome instability in all cancers is essential for development of therapeutics. The Kaposi's sarcoma-associated herpesvirus (KSHV) is an important human pathogen that causes multiple AIDS-related cancers. Recent studies have shown that during KSHV infection, cells show an increase in a double-strand DNA break marker, signifying a severe form of genome instability. Herein, we show that KSHV infection does cause DNA strand breaks. Moreover, we describe a novel molecular mechanism for genome instability involving the KSHV ORF57 protein interacting with the mRNA export complex, hTREX. We demonstrate that over-expression of ORF57 results in the formation of RNA:DNA hybrids, or R-loops, that lead to an increase in genome instability. DNA strand breaks have been previously reported in herpes simplex, cytomegalovirus and Epstein-Barr virus infected cells. Therefore, as this work describes for the first time the mechanism of R-loop induced genome instability involving a conserved herpesvirus protein, it may have far-reaching implications for other viral RNA export factors.

To investigate if specific exon 38 or 39 KMT2D missense variants (MVs) cause a condition distinct from Kabuki syndrome type 1 (KS1). Multiple individuals, with MVs in exons 38 or 39 of KMT2D that encode a highly conserved region of 54 amino acids flanked by Val3527 and Lys3583, were identified and phenotyped. Functional tests were performed to study their pathogenicity and understand the disease mechanism. The consistent clinical features of the affected individuals, from seven unrelated families, included choanal atresia, athelia or hypoplastic nipples, branchial sinus abnormalities, neck pits, lacrimal duct anomalies, hearing loss, external ear malformations, and thyroid abnormalities. None of the individuals had intellectual disability. The frequency of clinical features, objective software-based facial analysis metrics, and genome-wide peripheral blood DNA methylation patterns in these patients were significantly different from that of KS1. Circular dichroism spectroscopy indicated that these MVs perturb KMT2D secondary structure through an increased disordered to ɑ-helical transition. KMT2D MVs located in a specific region spanning exons 38 and 39 and affecting highly conserved residues cause a novel multiple malformations syndrome distinct from KS1. Unlike KMT2D haploinsufficiency in KS1, these MVs likely result in disease through a dominant negative mechanism.

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Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies. Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), often fatal malignancies afflicting HIV-infected patients. Previous research has shown that blockade of the ubiquitin proteasome system (UPS, a normal quality control pathway that degrades cellular proteins) is able to kill KSHV-infected lymphoma cells. A large component of the UPS is made up by the protein family known as the cullin-RING ubiquitin ligases (CRLs), which are activated by NEDD8 (a process known as NEDDylation). Recently, an inhibitor of NEDDylation (MLN4924) was developed and is currently in clinical trials as an anti-cancer drug. As NEDDylation has not been investigated for many viruses, we used this to compound examine its importance in KSHV biology. Firstly we show that NEDDylation is essential for the viability of KSHV-infected lymphoma cells, and MLN4924 treatment killed these cells by blocking NF-κB activity (required for KSHV latency gene expression and KSHV-associated cancer). Furthermore, we show that NEDDylation is required for KSHV to replicate its genome, a critical step in the production of new virus particles. Therefore, this research has identified a novel molecular mechanism that governs KSHV replication. Furthermore, it demonstrates that NEDDylation is a viable target for the treatment of KSHV-associated malignancies.

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway. When invading host cells, herpes viruses highjack cellular components to allow them to replicate. It has been long recognized that each herpes virus has a specific signature adaptor protein which, among other functions, inserts viral mRNA into the cellular mRNA nuclear export pathway, enabling production of viral proteins by the host cell. This process has been extensively studied in vivo and in vitro, but despite many efforts, the molecular and structural mechanisms of key interactions between viral adaptors and cellular mRNA export factors have not been described. Here we present the first atomic-resolution structure of the key complex between the archetypal viral adaptor ICP27 (from Herpes simplex virus 1) and the cellular mRNA export factor REF, responsible for introducing viral mRNA into the cellular nuclear export pathway. We demonstrate that despite the absence of obvious sequence similarity, the adaptor protein ORF57 from a different herpes virus (Herpesvirus saimiri) binds REF in the same site and in a similar way. We have identified and studied amino acid residues responsible for REF recognition. Together the data provide the first molecular insight into how herpesviral signature proteins recognize cellular proteins, obtaining access to the cellular mRNA export machinery.