SARS-CoV-2 evolution balances conflicting roles of N protein phosphorylation.

All lineages of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, contain mutations between amino acids 199 and 205 in the nucleocapsid (N) protein that are associated with increased infectivity. The effects of these mutations have been difficult to determine because N protein contributes to both viral replication and viral particle assembly during infection. Here, we used single-cycle infection and virus-like particle assays to show that N protein phosphorylation has opposing effects on viral assembly and genome replication.

Assembly of SARS-CoV-2 ribonucleosomes by truncated N variant of the nucleocapsid protein.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compacts the RNA genome into viral ribonucleoprotein (vRNP) complexes within virions. Assembly of vRNPs is inhibited by phosphorylation of the N protein serine/arginine (SR) region. Several SARS-CoV-2 variants of concern carry N protein mutations that reduce phosphorylation and enhance the efficiency of viral packaging.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 is responsible for compaction of the ∼30-kb RNA genome in the ∼90-nm virion. Previous studies suggest that each virion contains 35 to 40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex.

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation.

The nucleocapsid (N) protein of coronaviruses is responsible for compaction of the ∼30-kb RNA genome in the ∼100-nm virion. Cryo-electron tomography suggests that each virion contains 35-40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle, together with variations in substrate docking to sites on associated cyclin and Cks subunits. Many substrates are modified at multiple sites to provide more complex regulation.

HP1 proteins compact DNA into mechanically and positionally stable phase separated domains.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories.

Phosphoregulation of Phase Separation by the SARS-CoV-2 N Protein Suggests a Biophysical Basis for its Dual Functions.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder.

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell . The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder . Phosphorylation of the central disordered region is required for normal viral genome transcription , which occurs in a cytoplasmic structure called the replication transcription complex (RTC) .

Binding to small ubiquitin-like modifier and the nucleolar protein Csm1 regulates substrate specificity of the Ulp2 protease.

Ulp1 and Ulp2, in the yeast , are the founding members of deSUMOylating enzymes. These enzymes remove mall biquitin-like difier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme.

Recruitment of a SUMO isopeptidase to rDNA stabilizes silencing complexes by opposing SUMO targeted ubiquitin ligase activity.

Post-translational modification by SUMO (small ubiquitin-like modifier) plays important but still poorly understood regulatory roles in eukaryotic cells, including as a signal for ubiquitination by SUMO targeted ubiquitin ligases (STUbLs). Here, we delineate the molecular mechanisms for SUMO-dependent control of ribosomal DNA (rDNA) silencing through the opposing actions of a STUbL (Slx5:Slx8) and a SUMO isopeptidase (Ulp2). We identify a conserved region in the Ulp2 C terminus that mediates its specificity for rDNA-associated proteins and show that this region binds directly to the rDNA-associated protein Csm1.

A fast slam approach to freehand 3-d ultrasound reconstruction for catheter ablation guidance in the left atrium.

We present a method for real-time, freehand 3D ultrasound (3D-US) reconstruction of moving anatomy, with specific application towards guiding the catheter ablation procedure in the left atrium. Using an intracardiac echo (ICE) catheter with a pose (position/orientation) sensor mounted to its tip, we continually mosaic 2D-ICE images of a left atrium phantom model to form a 3D-US volume. Our mosaicing strategy employs a probabilistic framework based on simultaneous localization and mapping (SLAM), a technique commonly used in mobile robotics for creating maps of unexplored environments.

A probabilistic framework for freehand 3D ultrasound reconstruction applied to catheter ablation guidance in the left atrium.

Introduction: The catheter ablation procedure is a minimally invasive surgery used to treat atrial fibrillation. Difficulty visualizing the catheter inside the left atrium anatomy has led to lengthy procedure times and limited success rates. In this paper, we present a set of algorithms for reconstructing 3D ultrasound data of the left atrium in real-time, with an emphasis on automatic tissue classification for improved clarity surrounding regions of interest.

Catheter localization in the left atrium using an outdated anatomic reference for guidance.

We present a method for registering real-time ultrasound of the left atrium to an outdated, anatomic surface mesh model, whose shape differs from that of the anatomy. Using an intracardiac echo (ICE) catheter with mounted 6DOF electromagnetic position/orientation sensor (EPS), we acquire images of the left atrium and determine where the ICE catheter must be positioned relative to the surface mesh to generate similar, "virtual" ICE images. Further, we use an affine warping model to infer how the shape of the surface mesh differs from that of the atrium.

An incremental method for registering electroanatomic mapping data to surface mesh models of the left atrium.

We present a method for registering position and orientation data collected from an electroanatomic mapping system (EMS) to a surface mesh based on segmented Computed Tomography (CT) or Magnetic Resonance (MR) images of the left atrium. Our algorithm is based on the Unscented Particle Filter (UPF) for stochastic state estimation. Using an intracardiac echo (ICE) ultrasound catheter with mounted mapping sensor, we acquire ultrasound images of the atrium from multiple configurations and iteratively determine the catheter's pose with respect to anatomy.