Biology subjects

White, R. E.

Publications and source records attributed to White, R. E..

8 recordsLinked to original sources

bioRxiv2025

Extended poly(A) tails are a shared feature of herpesvirus mRNAs

Poly(A) tails are present on most cellular and viral mRNAs, providing a platform for poly(A)-binding proteins that stimulate translation and regulate the deadenylation and stability of transcripts in the cytoplasm. Here we leverage nanopore direct RNA sequencing to analyse the distribution of poly(A) tail lengths on cellular and viral mRNAs across Herpesviridae and other DNA and RNA virus infections. We find that herpesvirus mRNA poly(A) tails are consistently longer than those on cellular and other viral transcripts, presenting a previously unrecognized yet widespread mechanism to advantage herpesviral gene expression. This contrasts with the templated poly(A) tails on coronavirus RNAs and those on cytoplasmically transcribed poxviral mRNAs, which are more similar in length to those on host mRNAs. Herpesviral noncoding RNAs display differential poly(A) tailing patterns which do not correlate with nuclear localisation while individual herpesviral mRNAs also show variation in the extent to which their poly(A) tail lengths change during the virus lifecycle, suggestive of additional uncharacterised layers of poly(A) tail length regulation. Importantly, while we detect non-adenosine nucleotides within herpesviral poly(A) tails, which are known to oppose deadenylase activity, this "mixed tailing" is not at sufficient frequency to explain the widespread extended tails of herpesvirus mRNAs.

microbiology↗

bioRxiv2025

A direct RNA-seq-based EBV Latency Transcriptome Offers Insights into the Biogenesis of EBV Gene Products.

Epstein-Barr virus (EBV) ubiquitously infects humans, establishing lifelong persistence in B cells. In vitro, EBV-infected B cells can establish a lymphoblastoid cell line (LCL). EBVs transcripts in LCLs (Latency III) produce six nuclear proteins (EBNAs), two latency membrane proteins (LMPs) and various microRNAs and putative long non-coding RNAs (BARTs). The BART and EBNA transcription units are characterised by extensive alternative splicing. We generated LCLs with B95-8 EBV-BACs, including one engineered with "barcodes" in the first and last repeat of internal repeat 1 (IR1), and analysed their EBV transcriptomes using long-read nanopore direct RNA-seq. Our pipeline ensures appropriate mapping of the W promoter (Wp) 5 exon, and corrects W1-W2 exon counts that misalign to IR1. This suggests that splicing across IR1 largely includes all W exons, and that Wp-derived transcripts more frequently encode the EBNA-LP start codon than Cp transcripts. Analysis identified a short variant of exon W2 and a novel polyA site before EBNA2, provided insights into BHRF1 miRNA processing and suggested co-ordination between polyA and splice site usage, although improved read depth and integrity are required to confirm this. The BAC region disrupts the integrity of BART transcripts through premature polyadenylation and cryptic splice sites in the hygromycin expression cassette. Finally, a few transcripts extended across established gene boundaries, running from EBNA, to BART to LMP2 gene regions, sometimes including novel exons between EBNA1 and the BART promoter. We have produced an EBV annotation based on these findings to help others better characterise EBV transcriptomes in future. Data SummaryScripts (and the shell scripts used to combine commands into the pipeline), and guidance in their usage are available on Github (github.com/robertewhite/ebv-transcriptomics-tools). All of the RNA-seq raw data and analyses relevant to this study are available from the EMBL Nucleotide Archive Study accession PRJEB83447 (read files ERR14129300-303), or from the authors website, ebv.org.uk. Processed and analysed data is presented in Excel format in Supplementary tables ST1-8, and the intermediate data processing conducted in excel is linked from the front page of ebv.org.uk, alongside the scripts, raw reads and updated B95-8-BAC and prototype EBV transcriptome annotation (gff3) files. Impact statementThis article showcases the potential of direct RNA-seq to characterise complex transcriptomes like Epstein-Barr virus, highlights specific challenges of interpreting RNA-seq data, and presents tools to solve specific challenges of mapping RNA-seq reads to EBV exons. Biologically, the data analysis identifies several new aspects of Epstein-Barr virus transcription, offering insights into miRNA proteins, and identifies idiosyncrasies of the transcriptome of the widely used B95-8 BAC that will inform studies using this system. Finally we provide an improved annotation for EBV RNA-seq studies.

microbiology↗

bioRxiv2024

Sp140L Is a Novel Herpesvirus Restriction Factor

Herpesviruses, including Epstein-Barr Virus (EBV) - a human oncogenic viruses and essential trigger of multiple sclerosis, must bypass host DNA sensing mechanisms to establish lifelong, latent infection. Therefore, herpesviruses encode viral proteins to disrupt key host factors involved in DNA sensing and viral restriction. The first viral latency protein expressed, EBNA-LP, is essential for transformation of naive B cells and establishment of viral gene expression, yet its role in evading host defenses remains unclear. Using single-cell RNA sequencing of EBNA-LP-Knockout (LPKO)- infected B cells, we reveal an antiviral response landscape implicating the speckled proteins as key cellular restriction factors countered by EBNA-LP. Specifically, loss of SP100 or the primate-specific SP140L reverses the restriction of LPKO, suppresses a subset of canonically interferon-stimulated genes, and restores transcription of essential latent viral genes and cellular proliferation. Notably, we also identify Sp140L as a restriction target of the herpesvirus saimiri ORF3 protein, implying a role for Sp140L in immunity to other diverse DNA viruses. This study reveals Sp140L as a restriction factor that we propose links sensing and transcriptional suppression of viral DNA to an IFN-independent innate immune response, likely relevant to all nuclear DNA viruses. Significance StatementHerpesviruses, including the oncogenic Epstein-Barr virus (EBV), are restricted by DNA sensing during initial infection and therefore encode viral proteins to antagonize key restriction factors. We found that the EBV latency protein EBNA-LP, disrupts the speckled proteins Sp100 and Sp140L - an evolutionarily recent protein with unknown function, which we find promotes an anti-viral state that suppresses cellular proliferation, characterized by high induction of cellular anti-viral genes and suppressed transcription of essential viral latency genes. Sp140L also restricts the herpesvirus saimiri, which we find antagonizes Sp140L through the viral protein ORF3. Our study therefore identifies Sp140L as a novel restriction factor of diverse herpesviruses, and likely all DNA viruses, during a critical stage of initial viral infection.

microbiology↗

bioRxiv2024

Virus-induced paraspeckle-like condensates are essential hubs for gene expression and their formation drives genomic instability

The nucleus is a highly structured environment containing multiple membrane-less bodies formed through liquid-liquid phase separation. These provide spatial separation and concentration of specific biomolecules enabling efficient and discrete processes to occur which regulate gene expression. One such nuclear body, paraspeckles, are comprised of multiple paraspeckle proteins (PSPs) built around the architectural RNA, NEAT1_2. Paraspeckle function is yet to be fully elucidated but has been implicated in a variety of developmental and disease scenarios. We demonstrate that Kaposis sarcoma-associated herpesvirus (KSHV) drives formation of structurally distinct paraspeckles with a dramatically increased size and altered protein composition that are essential for productive lytic replication. We highlight these virus-induced paraspeckle-like structures form adjacent to virus replication centres, functioning as RNA processing hubs for both viral and cellular transcripts during infection. Notably, we reveal that PSP sequestration into virus-induced paraspeckle-like structures results in increased genome instability during both KSHV and Epstein Barr virus (EBV) infection, implicating their formation in virus-mediated tumorigenesis.

microbiology↗

bioRxiv2024

Epstein-Barr virus protein EBNA-LP engages YY1 through leucine-rich motifs to promote naïve B cell transformation

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro, EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naive but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2, EBNA-LP Knock Out (LPKO) virus-infected cells express EBNA2-activated genes efficiently. Therefore, a gap in knowledge exists as to what roles EBNA-LP plays in naive B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naive B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naive B cell outgrowth. However, we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naive and cord blood B cells. Because cellular PPAR-{gamma} coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1, a key regulator of DNA looping and metabolism, we examined the role of EBNA-LP in engaging cellular transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data and confirmed their biochemical association in LCLs by endogenous co-immunoprecipitation. Moreover, we found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. Finally, we used Cas9 to knockout YY1 in primary total B cells and naive B cells prior to EBV infection and found YY1 to be essential for EBV-mediated transformation. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naive B cells. Author SummaryEpstein-Barr Virus (EBV) is associated with various B cell lymphomas, particularly in immunosuppressed individuals. In the absence of a functional immune system, viral latency proteins, including EBV Nuclear Antigens (EBNAs) act as oncoproteins to promote tumorigenesis. EBNA-LP is one of the first viral proteins produced after infection and is important for the transformation of naive B cells. However, the roles of EBNA-LP during infection are largely undefined. In this study, developed an assay in which the role of wild type and mutant EBNA-LP could be investigated in context of primary naive B cells infected with an EBNA-LP Knock Out virus. Using this assay, we identified highly conserved leucine-rich motifs within EBNA-LP that are important for transformation of EBV-infected naive B cells. These conserved motifs associate with the cellular transcription factor YY1, an important transcriptional regulator in B cell development and in many cancers, that we now show is essential for outgrowth of EBV infected B cells. Our study provides further insights into the mechanisms by which EBV transforms naive B cells.

microbiology↗

bioRxiv2023

Saracatinib synergizes with enzalutamide to downregulate androgen receptor activity in castration resistant prostate cancer

Prostate cancer (PCa) remains the most diagnosed non-skin cancer amongst the American male population. Treatment for localized prostate cancer consists of androgen deprivation therapies (ADTs), which typically inhibit androgen production and the androgen receptor (AR). Though initially effective, a subset of patients will develop resistance to ADTs and the tumors will transition to castration-resistant prostate cancer (CRPC). Second generation hormonal therapies such as abiraterone acetate and enzalutamide are typically given to men with CRPC. However, these treatments are not curative and typically prolong survival only by a few months. Several resistance mechanisms contribute to this lack of efficacy such as the emergence of AR mutations, AR amplification, lineage plasticity, AR splice variants (AR-Vs) and increased kinase signaling. Having identified SRC kinase as a key tyrosine kinase enriched in CRPC patient tumors from our previous work, we evaluated whether inhibition of SRC kinase synergizes with enzalutamide or chemotherapy in several prostate cancer cell lines expressing variable AR isoforms. We observed robust synergy between the SRC kinase inhibitor, saracatinib, and enzalutamide, in the AR-FL+/AR-V+ CRPC cell lines, LNCaP95 and 22Rv1. We also observed that saracatinib significantly decreases AR Y534 phosphorylation, a key SRC kinase substrate residue, on AR-FL and AR-Vs, along with the AR regulome, supporting key mechanisms of synergy with enzalutamide. Lastly, we also found that the saracatinib-enzalutamide combination reduced DNA replication compared to the saracatinib-docetaxel combination, resulting in marked increased apoptosis. By elucidating this combination strategy, we provide pre-clinical data that suggests combining SRC kinase inhibitors with enzalutamide in select patients that express both AR-FL and AR-Vs.

cancer biology↗

bioRxiv2021

Massively parallel, computationally-guided design of a pro-enzyme

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to re-design any chosen enzyme to be similarly stimulus-responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for pro-enzyme design. As a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effective designed variants shows that they are inhibited by approximately 80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro-and catalytic domains based on the design models increases the degree of inhibition to 98%, but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing pro-enzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the pro-domain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of pro-proteins with precise spatial regulation. SignificanceProteins have shown promise as therapeutics and diagnostics, but their effectiveness is limited by our inability to spatially target their activity. To overcome this limitation, we developed a computationally-guided method to design inactive "pro-enzymes" or "zymogens," which are activated through cleavage by a protease. Since proteases are differentially expressed in various tissues and disease states, including cancer, these pro-enzymes could be targeted to the desired microenvironment. We tested our method on the therapeutically-relevant protein, carboxypeptidase G2 (CPG2). We designed Pro-CPG2s that are inhibited by 80-98% and are partially to fully re-activatable following protease treatment. The developed methodology, with further refinements, could pave the way for routinely designing protease-activated protein-based therapeutics and diagnostics that act in a spatially controlled manner.

biophysics↗

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↗