Two FAM134B isoforms differentially regulate ER dynamics during myogenesis.

Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated.

The inactivation of the Niemann Pick C1 cholesterol transporter restricts SARS-CoV-2 entry into host cells by decreasing ACE2 abundance at the plasma membrane.

Background: The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) that is involved in the mobilization of endocytosed cholesterol. Loss-of-function mutations in the NPC1 gene lead to the accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect both the integrity of lipid rafts and the endocytic pathway.

Quinoin, type 1 ribosome inactivating protein alters SARS-CoV-2 viral replication organelle restricting viral replication and spread.

SARS-CoV-2 pandemic clearly demonstrated the lack of preparation against novel and emerging viral diseases. This prompted an enormous effort to identify antivirals to curb viral spread and counteract future pandemics. Ribosome Inactivating Proteins (RIPs) and Ribotoxin-Like Proteins (RL-Ps) are toxin enzymes isolated from edible plants and mushrooms, both able to inactivate protein biosynthesis.

Identification of host dependency factors involved in SARS-CoV-2 replication organelle formation through proteomics and ultrastructural analysis.

Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection.

Electron microscopy: The key to resolve RNA viruses replication organelles.

Positive-sense single-stranded RNA viruses significantly reshape intracellular membranes to generate viral replication organelles that form a controlled niche in which nucleic acids, enzymes, and cofactors accumulate to assure an efficient replication of the viral genome. In recent years, advancements in electron microscopy (EM) techniques have enabled imaging of these viral factories in a near-native state providing significantly higher molecular details that have led to progress in our general understanding of virus biology. In this review, we describe the contribution of the cutting-edge EM approaches to the current knowledge of replication organelles biogenesis, structure, and functions.

Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remodels the endoplasmic reticulum (ER) to form replication organelles, leading to ER stress and unfolded protein response (UPR). However, the role of specific UPR pathways in infection remains unclear. Here, we found that SARS-CoV-2 infection causes marginal activation of signaling sensor IRE1α leading to its phosphorylation, clustering in the form of dense ER-membrane rearrangements with embedded membrane openings, and XBP1 splicing.

Human immunoglobulins are transported to HCMV viral envelope by viral Fc gamma receptors-dependent and independent mechanisms.

Human cytomegaloviruses (HCMVs) employ many different mechanisms to escape and subvert the host immune system, including expression of the viral IgG Fcγ receptors (vFcγRs) (gp34), (gp95), (gpRL13), and (gp68) gene products. The role of vFcγRs in HCMV pathogenesis has been reported to operate in infected cells by interfering with IgG-mediated effector functions. We found that gp34 and gp68 are envelope proteins that bind and internalize human IgGs on the surface of infected cells.

N-Phenylpyridine-3-Carboxamide and 6-Acetyl-1H-Indazole Inhibit the RNA Replication Step of the Dengue Virus Life Cycle.

Dengue virus (DENV) is a that causes the most prevalent arthropod-borne viral disease. Clinical manifestation of DENV infection ranges from asymptomatic to severe symptoms that can lead to death. Unfortunately, no antiviral treatments against DENV are currently available.

Membrane architects: how positive-strand RNA viruses restructure the cell.

Virus infection is a process that requires combined contributions from both virus and host factors. For this process to be efficient within the crowded host environment, viruses have evolved ways to manipulate and reorganize host structures to produce cellular microenvironments. Positive-strand RNA virus replication and assembly occurs in association with cytoplasmic membranes, causing a reorganization of these membranes to create microenvironments that support viral processes.

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication and Decreases Viral Titers and Disease Progression in Syrian Hamsters.

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication.

A protocol for full-rotation soft X-ray tomography of single cells.

The protocol describes step-by-step sample preparation, data acquisition, and segmentation of cellular organelles with soft X-ray tomography. It is designed for microscopes built to perform full-rotation data acquisition on specimens in cylindrical sample holders, such as the XM-2 microscope at the Advanced Light Source, LBNL; however, it might be generalized for similar sample holder designs for both synchrotron and table-top microscopes. For complete details on the use and execution of this profile, please refer to Loconte et al.

SARS-CoV-2 variants of concern display enhanced intrinsic pathogenic properties and expanded organ tropism in mouse models.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation.

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB.

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to long-lasting lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses, which can cause systemic complications.

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy.

Using soft X-ray tomography for rapid whole-cell quantitative imaging of SARS-CoV-2-infected cells.

High-resolution and rapid imaging of host cell ultrastructure can generate insights toward viral disease mechanism, for example for a severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Here, we employ full-rotation soft X-ray tomography (SXT) to examine organelle remodeling induced by SARS-CoV-2 at the whole-cell level with high spatial resolution and throughput. Most of the current SXT systems suffer from a restricted field of view due to use of flat sample supports and artifacts due to missing data.

The Biogenesis of Dengue Virus Replication Organelles Requires the ATPase Activity of Valosin-Containing Protein.

The dengue virus (DENV) causes the most prevalent arthropod-borne viral disease worldwide. While its incidence is increasing in many countries, there is no approved antiviral therapy currently available. In infected cells, the DENV induces extensive morphological alterations of the endoplasmic reticulum (ER) to generate viral replication organelles (vRO), which include convoluted membranes (CM) and vesicle packets (VP) hosting viral RNA replication.

Determinants in Nonstructural Protein 4A of Dengue Virus Required for RNA Replication and Replication Organelle Biogenesis.

Dengue virus (DENV) constitutes one of the most important arboviral pathogens affecting humans. The high prevalence of DENV infections, which cause more than 20,000 deaths annually, and the lack of effective vaccines or direct-acting antiviral drugs make it a global health concern. DENV genome replication occurs in close association with the host endomembrane system, which is remodeled to form the viral replication organelle that originates from endoplasmic reticulum (ER) membranes.

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 coronavirus disease 2019 (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.

Advanced microscopy technologies enable rapid response to SARS-CoV-2 pandemic.

The ongoing SARS-CoV-2 pandemic with over 80 million infections and more than a million deaths worldwide represents the worst global health crisis of the 21th century. Beyond the health crisis, the disruptions caused by the COVID-19 pandemic have serious global socio-economic consequences. It has also placed a significant pressure on the scientific community to understand the virus and its pathophysiology and rapidly provide anti-viral treatments and procedures in order to help the society and stop the virus spread.

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19.

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both M and PL proteases. These efforts identified a small number of hits for the M protease and no viable hits for the PL protease.

Microscopy-based assay for semi-quantitative detection of SARS-CoV-2 specific antibodies in human sera: A semi-quantitative, high throughput, microscopy-based assay expands existing approaches to measure SARS-CoV-2 specific antibody levels in human sera.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays.

Replication-Independent Generation and Morphological Analysis of Flavivirus Replication Organelles.

Positive-strand RNA viruses replicate in distinct membranous structures called replication organelles (ROs). Mechanistic studies of RO formation have been difficult because perturbations affecting viral replication have an impact on viral protein amounts, thus affecting RO biogenesis. Here, we present a detailed guide on how to use a replication-independent expression system, designated pIRO (plasmid-induced replication organelle formation), inducing bona fide flavivirus ROs in transfected cells.