Sally M. Harrison

Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway

ABSTRACT The Kaposis sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Cell Cycle Dependent Nucleolar Localization of the Coronavirus Nucleocapsid Protein

The nucleolus is a dynamic sub-nuclear structure which is involved in ribosome subunit biogenesis, modulation of cell growth and response to cell stress. The nucleolar proteome varies particularly with regard to the cell cycle. Viral proteins can localise to the nucleolus and using the coronavirus nucleocapsid (N) protein as a model, the cell cycle dependent trafficking of viral proteins to the nucleolus was investigated. Cell synchronisation studies coupled to live cell confocal microscopy indicated that nucleolar localisation of N protein was greater in the G2/M phase of the cell cycle than at other stages. This result was reinforced when FRAP and FLIP analysis indicated that N protein was more mobile within the nucleoplasm and nucleolus in the G2/M phase of the cell cycle. The data suggested that viral nucleolar proteins can also localise to the nucleolus in a cell cycle dependent manner and this may be related to dynamic trafficking.

Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture

The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry that, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination, which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross-strain immunity. Effective antiviral agents may therefore be advantageous in the treatment of IBV. Lithium chloride (LiCl) is a potent inhibitor of the DNA virus herpes simplex virus but not RNA viruses. The effect of LiCl on the replication of IBV was examined in cell culture using two model cell types; Vero cells, an African Green monkey kidney-derived epithelial cell line; and DF-1 cells, an immortalized chicken embryo fibroblast cell line. When treated with a range of LiCl concentrations, IBV RNA and protein levels and viral progeny production were reduced in a dose-dependent manner in both cell types, and the data indicated that inhibition was a cellular rather than a virucidal effect. Host cell protein synthesis still took place in LiCl-treated cells and the level of a standard cellular housekeeping protein remained unchanged, indicating that the effect of LiCl was specifically against IBV.

Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta and cellular HMGB1 proteins synergistically transactivate the KSHV ORF50 promoter

Kaposis sarcoma‐associated herpesvirus ‘replication transcriptional activator’ (Rta) plays a critical role in the switch from latency to lytic replication. Rta upregulates several lytic KSHV genes, including its own, through multiple mechanisms. We demonstrate that cellular HMGB1 binds and synergistically upregulates the ORF50 promoter in conjunction with Rta. No direct interaction between Rta and HMGB1 was observed, however a ternary complex is formed in the presence of Oct1. Furthermore, deletion of an Oct‐1 binding site within the ORF50 promoter ablates the HMGB1‐mediated synergistic response. These results suggest Rta autostimulation may be mediated by a transient complex involving Oct1 and HMGB1.

Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant‐Filtering Interface

Targeted hybridization enrichment prior to next‐generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the methods diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer‐predisposing genes or (2) whole‐exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome‐based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Characterization of the Nuclear Export Signal in the Coronavirus Infectious Bronchitis Virus Nucleocapsid Protein

ABSTRACT The nucleocapsid (N) protein of infectious bronchitis virus (IBV) localizes to the cytoplasm and nucleolus and contains an eight-amino-acid nucleolar retention motif. In this study, a leucine-rich nuclear export signal (NES) (291-LQLDGLHL-298) present in the C-terminal region of the IBV N protein was analyzed by using alanine substitution and deletion mutagenesis to investigate the relative contributions that leucine residues make to nuclear export and where these residues are located on the structure of the IBV N protein. The analysis indicated that Leu296 and Leu298 are required for efficient nuclear export of the protein. Structural information indicated that both of these amino acids are available for interaction with protein complexes involved in this process. However, export of N protein from the nucleus/nucleolus was not inhibited by leptomycin B treatment, indicating that N protein nuclear export is independent of the CRM1-mediated export pathway.

Diagnostic whole genome sequencing and split‐read mapping for nucleotide resolution breakpoint identification in CNTNAP2 deficiency syndrome

Whole genome sequencing (WGS) has the potential to report on all types of genetic abnormality, thus converging diagnostic testing on a single methodology. Although WGS at sufficient depth for robust detection of point mutations is still some way from being affordable for diagnostic purposes, low‐coverage WGS is already an excellent method for detecting copy number variants (“CNVseq”). We report on a family in which individuals presented with a presumed autosomal recessive syndrome of severe intellectual disability and epilepsy. Array comparative genomic hybridization (CGH) analysis had revealed a homozygous deletion apparently lying within intron 3 of CNTNAP2. Since this was too small for confirmation by FISH, CNVseq was used, refining the extent of this mutation to approximately 76.8 kb, encompassing CNTNAP2 exon 3 (an out‐of‐frame deletion). To characterize the precise breakpoints and provide a rapid molecular diagnostic test, we resequenced the CNVseq library at medium coverage and performed split read mapping. This yielded information for a multiplex polymerase chain reaction (PCR) assay, used for cascade screening and/or prenatal diagnosis in this family. This example demonstrates a rapid, low‐cost approach to converting molecular cytogenetic findings into robust PCR‐based tests.

Characterisation of cyclin D1 down-regulation in coronavirus infected cells

The positive strand RNA coronavirus, infectious bronchitis virus (IBV), induces a G2/M phase arrest and reduction in the G1 and G1/S phase transition regulator cyclin D1. Quantitative real‐time RT‐PCR and Western blot analysis demonstrated that cyclin D1 was reduced post‐transcriptionally within infected cells independently of the cell‐cycle stage at the time of infection. Confocal microscopy revealed that cyclin D1 decreased in IBV‐infected cells as infection progressed and inhibition studies indicated that a population of cyclin D1 could be targeted for degradation by a virus mediated pathway. In contrast to the SARS‐coronavirus, IBV nucleocapsid protein did not interact with cyclin D1.

OVA: integrating molecular and physical phenotype data from multiple biomedical domain ontologies with variant filtering for enhanced variant prioritization.

Motivation: Exome sequencing has become a de facto standard method for Mendelian disease gene discovery in recent years, yet identifying disease-causing mutations among thousands of candidate variants remains a non-trivial task. Results: Here we describe a new variant prioritization tool, OVA (ontology variant analysis), in which user-provided phenotypic information is exploited to infer deeper biological context. OVA combines a knowledge-based approach with a variant-filtering framework. It reduces the number of candidate variants by considering genotype and predicted effect on protein sequence, and scores the remainder on biological relevance to the query phenotype. We take advantage of several ontologies in order to bridge knowledge across multiple biomedical domains and facilitate computational analysis of annotations pertaining to genes, diseases, phenotypes, tissues and pathways. In this way, OVA combines information regarding molecular and physical phenotypes and integrates both human and model organism data to effectively prioritize variants. By assessing performance on both known and novel disease mutations, we show that OVA performs biologically meaningful candidate variant prioritization and can be more accurate than another recently published candidate variant prioritization tool. Availability and implementation: OVA is freely accessible at http://dna2.leeds.ac.uk:8080/OVA/index.jsp Supplementary information: Supplementary data are available at Bioinformatics online. Contact: umaan@leeds.ac.uk

Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target.

Significance By investigating cell surface proteins of Ewing sarcoma we have identified an antigen that is uniquely expressed on these tumor cells compared with mesenchymal stem cells. This protein acts as a target for antibody drug conjugates that are internalized and can kill these tumor cells, presaging translating to clinical use in treating Ewing sarcoma, especially metastatic disease. The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.