Biology subjects

Yewdell, J. W.

Publications and source records attributed to Yewdell, J. W..

At least 19 recordsLinked to original sources

bioRxiv2025

Nanoscopy Reveals Heparan Sulfate Clusters as Docking Sites for SARS-CoV-2 Attachment and Entry

Virus entry is thought to involve binding a unique receptor for cell attachment and cytosolic entry. For SARS-CoV-2 underlying the COVID-19 pandemic, angiotensin-converting enzyme 2 (ACE2) is widely considered the receptor for cell-surface attachment and cytosolic entry. Using advanced light microscopy to resolve individual virions and receptors, we found instead that heparan sulfate (HS), not ACE2, mediates SARS-CoV-2 cell-surface attachment and subsequent endocytosis. ACE2 functions only downstream of HS to enable viral genome expression. Instead of binding single HS molecules that electrostatically interact with viral surface proteins weakly, SARS-CoV-2 binds clusters of [~]6-137 HS molecules projecting 60-410 nm above the plasma membrane. These tall, HS-rich clusters, present at about one per 6 m{superscript 2}, act as docking sites for viral attachment. Blocking HS binding with the clinically used HS-binding agent pixantrone strongly inhibited an authentic pathogen, the SARS-CoV-2 Omicron JN.1 subvariant, from attaching to and infecting human airway cells. This work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting downstream, thereby identifying HS interactions as a key anti-COVID-19 strategy. This paradigm and its therapeutic implications may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator. Statement of SignificanceViral entry, a crucial antiviral target, is typically thought to involve binding its unique receptor for the cell surface attachment and subsequent entry. We examined this concept with advanced microscopies to resolve individual receptors and SARS-CoV-2 virions responsible for the COVID-19 pandemic. We discovered two receptors for viral entry: heparan sulfate, a polysaccharide that may bind many viruses, mediates viral attachment and subsequent endocytosis, whereas angiotensin-converting enzyme 2 (ACE2), the generally assumed SARS-CoV-2 receptor, acts only downstream to facilitate viral infection. This model suggests perturbation of HS binding as a more effective anti-COVID-19 strategy than previously recognized. It may apply broadly beyond COVID-19 because, analogous to SARS-CoV-2, HS binds many other viruses but is only considered an attachment regulator.

microbiology↗

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗

bioRxiv2024

Mitochondrial Hyperactivity and Reactive Oxygen Species Drive Innate Immunity to the Yellow Fever Virus-17D Live-Attenuated Vaccine

The yellow fever virus 17D (YFV-17D) live attenuated vaccine is considered one of the successful vaccines ever generated associated with high antiviral immunity, yet the signaling mechanisms that drive the response in infected cells are not understood. Here, we provide a molecular understanding of how metabolic stress and innate immune responses are linked to drive type I IFN expression in response to YFV-17D infection. Comparison of YFV-17D replication with its parental virus, YFV-Asibi, and a related dengue virus revealed that IFN expression requires RIG-I-like Receptor signaling through MAVS, as expected. However, YFV-17D uniquely induces mitochondrial respiration and major metabolic perturbations, including hyperactivation of electron transport to fuel ATP synthase. Mitochondrial hyperactivity generates reactive oxygen species (mROS) and peroxynitrite, blocking of which abrogated IFN expression in non-immune cells without reducing YFV-17D replication. Scavenging ROS in YFV-17D-infected human dendritic cells increased cell viability yet globally prevented expression of IFN signaling pathways. Thus, adaptation of YFV-17D for high growth uniquely imparts mitochondrial hyperactivity generating mROS and peroxynitrite as the critical messengers that convert a blunted IFN response into maximal activation of innate immunity essential for vaccine effectiveness.

immunology↗

bioRxiv2023

Syncytia Formation Promotes Virus Resistance to Interferon and Neutralizing Antibodies

SARS-CoV-2, like many viruses, generates syncytia but the role of syncytia formation in viral evolution remains unknown. Using SARS-CoV-2 and SARS-CoV-2 Spike (S) replacement vesicular stomatitis (VSV), we show that S-mediated syncytia impair the antiviral effects of interferons in cultured cells, human lung cell cultures, and hACE2 transgenic mice. Amino acid substitutions that modulate syncytia formation in Delta- and Omicron-encoded S have parallel effects on viral interferon resistance. S-mediated syncytia compromise antibody-mediated virus neutralization in cultured cells. We recapitulate interferon and neutralizing antibody resistance in syncytia generated by the orthoreovirus p14 fusion-associated small transmembrane (FAST) protein in VSV, influenza virus, and seasonal coronavirus OC43 infections. These findings explain selection of SARS-CoV-2 fusogenic variants in humans and, more generally, the evolution of fusogenic viruses driven by adaptive and innate immunity.

microbiology↗

bioRxiv2023

An "alert state" ribosome population acts as a master regulator of cytokine mediated processes

Inflammatory cytokines are pivotal to immune responses. Upon cytokine exposure, cells enter an "alert-state" that enhances their visibility to the immune system. Here, we identified an "alert-state" subpopulation of ribosomes (ASRs) defined by the presence of the P-stalk. We show that ASRs are formed in response to cytokines linked to tumor immunity, and are involved in the preferential translation of mRNAs vital for the cytokine response. Mechanistically, ASRs are required for the efficient translation of transmembrane domains of receptor molecules involved in cytokine-mediated processes. Importantly, loss of the ASR prevents CD8+ T cell recognition and killing, and inhibitory cytokines like TGF{beta} hinder ASR formation, suggesting that the ASR is a central regulatory hub upon which multiple signals converge. Thus, the ASR is an essential mediator of the cellular rewiring that occurs following cytokine exposure, via the translational regulation of this process.

immunology↗

bioRxiv2023

Cell Surface Nucleocapsid Protein Expression: A Betacoronavirus Immunomodulatory Strategy

We recently reported that SARS-CoV-2 Nucleocapsid (N) protein is abundantly expressed on the surface of both infected and neighboring uninfected cells, where it enables activation of Fc receptor-bearing immune cells with anti-N antibodies (Abs) and inhibits leukocyte chemotaxis by binding chemokines (CHKs). Here, we extend these findings to N from the seasonal human coronavirus (HCoV)-OC43, which is also robustly expressed on the surface of infected and non-infected cells by binding heparan-sulfate/heparin (HS/H). HCoV-OC43 N binds with high affinity to the same set of 11 human CHKs as SARS-CoV-2 N, but also to a non-overlapping set of 6 cytokines (CKs). As with SARS-CoV-2 N, HCoV-OC43 N inhibits CXCL12{beta}-mediated leukocyte migration in chemotaxis assays, as do all highly pathogenic and endemic HCoV N proteins. Together, our findings indicate that cell surface HCoV N plays important evolutionary conserved roles in manipulating host innate immunity and as a target for adaptive immunity.

microbiology↗

bioRxiv2022

Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence SummaryRare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

immunology↗

bioRxiv2022

NEDD8 activity is important for direct antigen MHC class I antigen presentation

Successful direct MHC class I antigen presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides which can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin-proteasome system, however other ubiquitin-like (UBL) proteins have also been implicated in protein degradation and direct antigen presentation. Here, we examine the role of neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) in direct antigen presentation. NEDD8 is the UBL with highest similarity to ubiquitin and fusion of NEDD8 to the amino-terminus of a target protein can lead to the target proteins degradation. We find that appending NEDD8 to the N-terminus of the model antigen ovalbumin resulted in degradation by both the proteasome and autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1 (NUB1), resulted in peptide presentation. When directly compare to ubiquitin, NEDD8-fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924, inhibited the presentation of peptides from Defective Ribosomal Products (DRiPs) derived from a model antigen. These results demonstrate that NEDD8 activity in the cell is important for direct antigen presentation, but not by directly targeting proteins for degradation.

immunology↗

bioRxiv2021

Cell Surface SARS-CoV-2 Nucleocapsid Protein Modulates Innate and Adaptive Immunity

SARS-CoV-2 nucleocapsid protein (N) induces strong antibody and T cell responses. Although considered to be localized in the cytosol, we readily detect N on the surface of live cells. N released by SARS-CoV-2 infected cells or N-expressing transfected cells binds to neighboring cells by electrostatic high-affinity binding to heparan sulfate and heparin, but not other sulfated glycosaminoglycans. N binds with high affinity to 11 human chemokines, including CXCL12{beta}, whose chemotaxis of leukocytes is inhibited by N from SARS-CoV-2, SARS-CoV-1, and MERS CoV. Anti-N Abs bound to the surface of N expressing cells activate Fc receptor-expressing cells. Our findings indicate that cell surface N manipulates innate immunity by sequestering chemokines and can be targeted by Fc expressing innate immune cells. This, in combination with its conserved antigenicity among human CoVs, advances its candidacy for vaccines that induce cross-reactive B and T cell immunity to SARS-CoV-2 variants and other human CoVs, including novel zoonotic strains.

microbiology↗

bioRxiv2021

Variations in cell-surface ACE2 levels alter direct binding of SARS-CoV-2 Spike protein and viral infectivity: Implications for measuring Spike protein interactions with animal ACE2 orthologs

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, the most severe pandemic in a century. The virus gains access to host cells when the viral Spike protein (S-protein) binds to the host cell-surface receptor angiotensin-converting enzyme 2 (ACE2). Studies have attempted to understand SARS-CoV-2 S-protein interaction with vertebrate orthologs of ACE2 by expressing ACE2 orthologs in mammalian cells and measuring viral infection or S-protein binding. Often these cells only transiently express ACE2 proteins and levels of ACE2 at the cell surface are not quantified. Here, we describe a cell-based assay that uses stably transfected cells expressing ACE2 proteins in a bi-cistronic vector with an easy to quantify reporter protein to normalize ACE2 expression. We found that both binding of the S-protein receptor-binding domain (RBD) and infection with a SARS-CoV-2 pseudovirus is proportional to the amount of human ACE2 expressed at the cell surface, which can be inferred by quantifying the level of reporter protein, Thy1.1. We also compared different ACE2 orthologs which were expressed in stably transfected cells expressing equivalent levels of Thy1.1. When ranked for either viral infectivity or RBD binding, mouse ACE2 had a weak to undetectable affinity for S-protein while human ACE2 was the highest level detected and feline ACE2 had an intermediate phenotype. The generation of stably transfected cells whose ACE2 level can be normalized for cross-ortholog comparisons allows us to create a reusable cellular library useful for measuring emerging SARS-CoV-2 variants ability to potentially infect different animals. ImportanceSARS-CoV-2 is a zoonotic virus responsible for the worst global pandemic in a century. An understanding of how the virus can infect other vertebrate species is important for controlling viral spread and understanding the natural history of the virus. Here we describe a method to generate cells stably expressing equivalent levels of different ACE2 orthologs, the receptor for SARS-CoV-2, on the surface of a human cell line. We find that both binding of the viral Spike protein receptor binding domain (RBD) and infection of cells with a SARS-CoV-2 pseudovirus are proportional to ACE2 levels at the cell surface. Adaptation of this method will allow for the creation of a library of stable transfected cells expressing equivalent levels of different vertebrate ACE2 orthologs which can be repeatedly used for identifying vertebrate species which may be susceptible to infection with SARS-CoV-2 and its many variants.

microbiology↗

bioRxiv2020

Single cell BCR and transcriptome analysis after respiratory virus infection reveals spatiotemporal dynamics of antigen-specific B cell responses.

B cell responses are a critical component of anti-viral immunity. However, a comprehensive picture of antigen-specific B cell responses, differentiation, clonal proliferation and dynamics in different organs after infection is lacking. Here, we combined single-cell RNA sequencing with single-cell B cell receptor (BCR) characterization of antigen-specific cells in the draining lymph nodes, spleen and lungs after influenza infection. We identify several novel B cell subpopulations forming after infection and find organ-specific differences that persist over the course of the response. We discover important transcriptional differences between memory cells in lungs and lymphoid organs and describe organ-restricted clonal expansion. Strikingly, by combining BCR mutational analysis, monoclonal antibody expression and affinity measurements we find no differences between germinal center (GC)-derived memory and plasmacells, at odds with an affinity-based selection model. By linking antigen-recognition with transcriptional programming, clonal-proliferation and differentiation, these finding provide important advances in our understanding of antiviral B cell immunity.

immunology↗

bioRxiv2020

Decoding mRNA translatability and stability from 5'UTR

Precise control of protein synthesis by engineering sequence elements in 5 untranslated region (5UTR) remains a fundamental challenge. To accelerate our understanding of cis-regulatory code embedded in 5UTR, we devised massively parallel reporter assays from a synthetic mRNA library composed of over one million 5UTR variants. A completely randomized 10-nucleotide sequence preceding an upstream open reading frame (uORF) and downstream GFP leads to a broad range of mRNA translatability and stability in mammalian cells. While efficient translation protects mRNA from degradation, uORF translation triggers mRNA decay in a UPF1-dependent manner. We also identified translational inhibitory elements in 5UTR with G-quadruplex as a mark for mRNA decay in the P-body. Unexpectedly, an unstructured A-rich element in 5UTR, while enabling cap-independent translation, destabilizes mRNAs in the absence of translation. Our results not only expose diverse sequence features of 5UTR in controlling mRNA translatability, but also reveal ribosome-dependent and -independent mRNA surveillance pathways.

biochemistry↗

bioRxiv2020

Genome-wide Screens Identify Lineage- and Tumor Specific-Genes Modulating MHC-I and MHC-II Immunosurveillance in Human Lymphomas

Tumors frequently subvert MHC class I (MHC-I) peptide presentation to evade CD8+ T cell immunosurveillance. To better define the regulatory networks controlling antigen presentation, we employed genome-wide screening in human diffuse large B cell lymphomas (DLBCLs). This approach revealed dozens of novel genes that positively and negatively modulate MHC-I cell surface levels. Identified genes cluster in multiple pathways including cytokine signaling, mRNA processing, endosomal trafficking, and protein metabolism. Many genes exhibit lymphoma subtype- or tumor-specific MHC-I regulation, and a majority of primary DLBCL tumors display genetic alterations in multiple regulators. We establish that the HSP90 co-chaperone SUGT1 is a major positive regulator of both MHC-I and MHC-II cell surface expression. Further, pharmacological inhibition of two negative regulators of antigen presentation, EZH2 and thymidylate synthase, enhances DLBCL MHC-I presentation. These and other genes represent potential targets for manipulating MHC-I immunosurveillance in cancers, infectious diseases, and autoimmunity.

cancer biology↗

bioRxiv2019

Discovery Of UFO Proteins: Human-Virus Chimeric Proteins Generated During Influenza Virus Infection

Influenza A virus (IAV) is a threat to mankind because it generates yearly epidemics and poorly predictable sporadic pandemics with catastrophic potential. IAV has a small RNA genome composed of 8 mini-chromosomes (segments) that constitute a 5UTR followed by a coding region and a 3UTR. Transcription of IAV RNA into mRNA depends on host mRNA, as the viral polymerase cleaves 5m7G-capped nascent transcripts to use as primers to initiate viral mRNA synthesis. We hypothesized that captured host transcripts bearing AUG could drive the expression of upstream ORFs in the viral segments, a phenomenon that would depend on the translatability of the viral 5UTRs. Here we report the existence of this mechanism, which generates host-virus chimeric proteins. We label these proteins as Upstream Flu ORFs (UFO). Depending on the frame, two types of host-virus UFO proteins are made: canonical viral proteins with human-derived N term extensions or novel uncharacterized proteins. Here we show that both types are made during IAV infection. Sequences that enable chimeric protein synthesis are conserved across IAV strains, indicating that selection allowed the expansion of the proteome diversity of IAV in infected cells to include multiple human-virus proteins.

microbiology↗

bioRxiv2018

Computational Discovery of Evolutionary Conserved Enterovirus 71 RNA Secondary Structures

Viral RNAs store information in their sequence and structure. Little, however is known about the contribution of RNA structure to viral replication and evolution. We previously described RNA ISRAEU to computationally identify structured regions in viral RNA based on evolutionary conservation of predicted structures. Here, we apply RNA GUESS (Generator of Uniformly Evolved Secondary Structures) to better understand enterovirus 71, a highly pathogenic human picornavirus. RNA GUESS identified previously known evolutionarily conserved picornavirus RNA structures, and uniquely predicted RNA structures with significant structural conservation despite the lack of an obvious sequence conservation. One structure consists of a stretch of obligatorily unpaired nucleotides that can potentially interact with RNA-binding proteins and siRNAs. Another, forms a potential RNA switch that can form two alternative structures while the third and fourth constitute hairpin-like structures. These findings provide a launching point for physical and genetic studies regarding the function and structures of these RNA elements.

bioinformatics↗

bioRxiv2018

Novel Computational Method to Define RNA PSRs Explains Influenza A Virus Nucleotide Conservation

ABSTRACTRNA molecules often fold into evolutionarily selected functional structures. Yet, the literature offers neither a satisfactory definition for \"structured RNA regions\", nor a computational method to accurately identify such regions. Here, we define structured RNA regions based on the premise that both stems and loops in functional RNA structures should be conserved among RNA molecules sharing high sequence homology. In addition, we present a computational approach to identify RNA regions possessing evolutionarily conserved secondary structures, RNA ISRAEU (RNA Identification of Structured Regions As Evolutionary Unchanged). Applying this method to H1N1 influenza mRNAs revealed previously unknown structured RNA regions that are potentially essential for viral replication and/or propagation. Evolutionary conservation of RNA structural elements may explain, in part, why mutations in some nucleotide positions within influenza mRNAs occur significantly more often than in others. We found that mutations occurring in conserved nucleotide positions may be more disruptive for structured RNA regions than single nucleotide polymorphisms in positions that are more prone to changes. Finally, we predicted computationally a previously unknown stem-loop structure and demonstrated that oligonucleotides complementing the stem (but not the loop or unrelated sequences) reduce viral replication in vitro. These results contribute to understanding influenza A virus evolution and can be applied to rational design of attenuated vaccines and/or drug designs based on disrupting conserved RNA structural elements.\n\nAUTHOR SUMMARYRNA structures play key biological roles. However, the literature offers neither a satisfactory definition for \"structured RNA regions\" nor the computational methodology to identify such regions. We define structured RNA regions based on the premise that functionally relevant RNA structures should be evolutionarily conserved, and devise a computational method to identify RNA regions possessing evolutionarily conserved secondary structural elements. Applying this method to influenza virus mRNAs of pandemic and seasonal H1N1 influenza A virus generated Predicted Structured Regions (PSRs), which were previously unknown. This explains the previously mysterious sequence conservation among evolving influenza strains. Also, we have experimentally supported existence of a computationally predicted stem-loop structure predicted computationally. Our approach may be useful in designing live attenuated influenza vaccines and/or anti-viral drugs based on disrupting necessary conserved RNA structures.

bioinformatics↗

bioRxiv2018

Neuraminidase Inhibition Contributes to Influenza A Virus Neutralization by Anti-Hemagglutinin Stem Antibodies

Broadly neutralizing antibodies (Abs) that bind the influenza virus hemagglutinin (HA) stem may enable universal influenza vaccination. Here, we show that anti-stem Abs sterically inhibit viral neuraminidase activity against large substrates, with activity inversely proportional to the length of the fibrous NA stalk that supports the enzymatic domain. By modulating NA stalk length in recombinant IAVs, we show that anti-stem Abs inhibit virus release from infected cells by blocking NA, accounting for their in vitro neutralization activity. NA inhibition contributes to anti-stem Ab protection in influenza infected mice, likely due at least in part to NA-mediated inhibition of Fc{gamma}R dependent activation of innate immune cells by antibody bound to virions. FDA approved NA inhibitors enhance anti-stem based Fc{gamma} dependent immune cell activation, raising the possibility of therapeutic synergy between NA inhibitors and anti-stem mAb treatment in humans.

microbiology↗

bioRxiv2018

Outflanking Immunodominance to Target Subdominant Broadly Neutralizing Epitopes

A major obstacle to vaccination to antigenically variable viruses is skewing of antibody responses to immunodominant epitopes. For influenza virus hemagglutinin (HA), the immunodominance of the variable head impairs responses to the highly conserved stem. Here, we show that head immunodominance depends on the physical attachment of head to stem. Stem immunogenicity is enhanced by immunizing with stem only-constructs or by increasing local HA concentration in the draining lymph node. Surprisingly, co-immunization of HA and stem alters stem-antibody class switching. Our findings delineate strategies for overcoming immunodominance with important implications for human vaccination.

immunology↗