The Kaposi's sarcoma-associated herpesvirus ORF57 protein and its multiple roles in mRNA biogenesis

Post-transcriptional events which regulate mRNA biogenesis are fundamental to the control of gene expression. A nascent mRNA is therefore steered through multimeric RNA–protein complexes that mediate its capping, splicing, polyadenylation, nuclear export, and ultimately its translation. Kaposi’s sarcoma-associated herpesvirus (KSHV) mRNA transport and accumulation protein, or ORF57, is a functionally conserved protein found in all herpesviruses which plays a pivotal role in enhancing viral gene expression at a post-transcriptional level. As such, ORF57 has been implicated in multiple steps of RNA biogenesis, including augmenting viral splicing, protecting viral RNAs from degradation to enhancing viral mRNA nuclear export and translation. In this review, we highlight the multiple roles of KSHV ORF57 in regulating the post-transcriptional events which are fundamental to the control of virus gene expression

Global analysis of RNA-binding protein dynamics by comparative and enhanced RNA interactome capture

Interactions between RNA-binding proteins (RBPs) and RNAs are critical to cell biology. However, methods to comprehensively and quantitatively assess these interactions within cells were lacking. RNA interactome capture (RIC) uses in vivo UV crosslinking, oligo(dT) capture, and proteomics to identify RNA-binding proteomes. Recent advances have empowered RIC to quantify RBP responses to biological cues such as metabolic imbalance or virus infection. Enhanced RIC exploits the stronger binding of locked nucleic acid (LNA)-containing oligo(dT) probes to poly(A) tails to maximize RNA capture selectivity and efficiency, profoundly improving signal-to-noise ratios. The subsequent analytical use of SILAC and TMT proteomic approaches, together with high-sensitivity sample preparation and tailored statistical data analysis, substantially improves RIC’s quantitative accuracy and reproducibility. This optimized approach is an extension of the original RIC protocol. It takes 3 d plus 2 weeks for proteomics and data analysis and will enable the study of RBP dynamics under different physiological and pathological conditions

Absolute quantitation of individual SARS-CoV-2 RNA molecules provides a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19

System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection

International audienceGraphical Abstract Highlights d A quarter of the RBPome changes upon SINV infection d Alterations in RBP activity are largely explained by changes in RNA availability d Altered RBPs are crucial for viral infection efficacy d GEMIN5 binds to the 5 0 end of SINV RNAs and regulates viral gene expressio

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

Functional analysis of the KSHV ORF57 protein in mRNA nuclear export mechanisms

Kaposi's sarcoma-associated Herpesvirus (KSHV; HHV -8) is associated with multiple malignancies, including Kaposi's sarcoma. KSHV has two distinct life cycle phases, latent persistence and lytic replication. In contrast to other oncogenic Herpesviruses, lytic replication plays an important part in tumourigenicity and pathogenesis of KSHV. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. This in turn may lead to novel therapies, which could become an important strategy for the treatment of KSHV -associated diseases. Post-transcriptional regulation of RNA biogenesis is fundamental to KSHV lytic gene expression. The KSHV ORF57 protein plays an essential role in viral RNA processing, transcription, splicing, mRNA stability, nuclear mRNA export and translation. To date, it is unknown how ORF57 co-ordinates these many roles. Functional diversity of a protein can be achieved by post-translational modifications. We demonstrate that ORF57 is post-translationally methylated and inhibition of methylation has a dramatic effect on the ability of ORF57 to export intronless viral RN A out of the nucleus. This work shows that hypomethylation of ORF57 enhances its ability to bind RNA. Attempts were made to find ORF57 residues which are methylated and results identified PRMT5, a cellular protein methyltransferase as well as the putative demethylase MINA, which interact with ORF57. Furthermore, a proteomic-based approach was used to identify ce~lular proteins which are changed in their intracellular distribution or abundance upon ORF57 expression. This SILAC-based approach highlighted proteins and pathways affected by ORF57. Data showed changes in RNA processing pathways previously unknown to be affected by ORF57, e.g. mRNA polyadenylation and nucleoskeleton rearrangements. In addition, changes of putative, novel components of the human TREX complex were found. Data presented will provide a valuable resource not only to enhance our understanding of this viral pathogen but also to show new routes for drug intervention.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Model of how sequestration of hTREX by ORF57 leads to R-loops and genome instability.

<p>In a healthy cell, components of hTREX are recruited to cellular pre-mRNA during the inter-linked processes of transcription and splicing. These components then act to stabilise the newly transcribed mRNA. In situations when hTREX is rendered non-functional through mutation or siRNA, the newly transcribed mRNA can become unstable and anneal to the template strand of DNA forming R-loops and leading to an increase in DNA strand breaks. During KSHV infection or exogenous ORF57 expression, ORF57 recruits the hTREX complex, essentially replicating a system of mutated hTREX and leading to an increase in genome instability.</p

Chromosome instability in ORF57 expressing cells.

<p>(<b>A</b>) iORF57 293 cells remained uninduced or induced for 24 hours, then fixed on coverslips and DNA-stained with DAPI. Confocal microscopy was used to identify and image mitotic cells. Three representative images are shown for uninduced and induced cells and merged images include the phase contrast image. Chromosome lagging in the induced samples is highlighted by white arrows. (<b>B</b>) HEK 293T cells mock transfected or transfected with EGFP or EGFP-ORF57 were fixed onto coverslips and stained with DAPI for imaging of mitotic cells. Chromosome lagging in the EGFP-ORF57 expressing cell is highlighted by a white arrow.</p