Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19

Quantitative Proteomic Analysis Reveals apoE4-Dependent Phosphorylation of the Actin-Regulating Protein VASP

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease. While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here, we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in vasodilator-stimulated phosphoprotein (VASP) S235 phosphorylation in a protein kinase A (PKA)-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects

LOS TENTÁCULOS DE LA MUERTE CORONAVIRUS CREA PUENTES ENTRE LAS CÉLULAS INFECTADAS

En un estudio publicado en la prestigiosa revista científica Cell, mediante unas magníficas imágenes por microscopia electrónica, se pudo demostrar que el coronavirus puede transformar a la célula infectada, comportándose como un arquitecto que logra cambiar la estructura celular, contribuyendo así a la propagación de la infección a través de las células. Estos descubrimientos permiten obtener algunos puntos claves para la comprensión de la propagación de este virus mortal

Resistance to ATR Inhibitors Is Mediated by Loss of the Nonsense-Mediated Decay Factor UPF2.

Over one million cases of gastric cancer are diagnosed each year globally, and the metastatic disease continues to have a poor prognosis. A significant proportion of gastric tumors have defects in the DNA damage response pathway, creating therapeutic opportunities through synthetic lethal approaches. Several small-molecule inhibitors of ATR, a key regulator of the DNA damage response, are now in clinical development as targeted agents for gastric cancer. Here, we performed a large-scale CRISPR interference screen to discover genetic determinants of response and resistance to ATR inhibitors (ATRi) in gastric cancer cells. Among the top hits identified as mediators of ATRi response were UPF2 and other components of the nonsense-mediated decay (NMD) pathway. Loss of UPF2 caused ATRi resistance across multiple gastric cancer cell lines. Global proteomic, phosphoproteomic, and transcriptional profiling experiments revealed that cell-cycle progression and DNA damage responses were altered in UPF2-mutant cells. Further studies demonstrated that UPF2-depleted cells failed to accumulate in G1 following treatment with ATRi. UPF2 loss also reduced transcription-replication collisions, which has previously been associated with ATRi response, thereby suggesting a possible mechanism of resistance. Our results uncover a novel role for NMD factors in modulating response to ATRi in gastric cancer, highlighting a previously unknown mechanism of resistance that may inform the clinical use of these drugs.SignificanceLoss of NMD proteins promotes resistance to ATR inhibitors in gastric cancer cells, which may provide a combination of therapeutic targets and biomarkers to improve the clinical utility of these drugs

Brahma safeguards canalization of cardiac mesoderm differentiation

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory

The Global Phosphorylation Landscape of SARS-CoV-2 Infection

International audienceThe causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies