Unbiased discovery of autoantibodies associated with severe COVID-19 via genome-scale self-assembled DNA-barcoded protein libraries.

Pathogenic autoreactive antibodies that may be associated with life-threatening coronavirus disease 2019 (COVID-19) remain to be identified. Here, we show that self-assembled genome-scale libraries of full-length proteins covalently coupled to unique DNA barcodes for analysis by sequencing can be used for the unbiased identification of autoreactive antibodies in plasma samples. By screening 11,076 DNA-barcoded proteins expressed from a sequence-verified human ORFeome library, the method, which we named MIPSA (for Molecular Indexing of Proteins by Self-Assembly), allowed us to detect circulating neutralizing type-I and type-III interferon (IFN) autoantibodies in five plasma samples from 55 patients with life-threatening COVID-19. In addition to identifying neutralizing type-I IFN-α and IFN-ω autoantibodies and other previously known autoreactive antibodies in patient plasma, MIPSA enabled the detection of as yet unidentified neutralizing type-III anti-IFN-λ3 autoantibodies that were not seen in healthy plasma samples or in convalescent plasma from ten non-hospitalized individuals with COVID-19. The low cost and simple workflow of MIPSA will facilitate unbiased high-throughput analyses of protein-antibody, protein-protein and protein-small-molecule interactions.

Neutralizing IFNL3 Autoantibodies in Severe COVID-19 Identified Using Molecular Indexing of Proteins by Self-Assembly.

Unbiased antibody profiling can identify the targets of an immune reaction. A number of likely pathogenic autoreactive antibodies have been associated with life-threatening SARS-CoV-2 infection; yet, many additional autoantibodies likely remain unknown. Here we present Molecular Indexing of Proteins by Self Assembly (MIPSA), a technique that produces ORFeome-scale libraries of proteins covalently coupled to uniquely identifying DNA barcodes for analysis by sequencing. We used MIPSA to profile circulating autoantibodies from 55 patients with severe COVID-19 against 11,076 DNA-barcoded proteins of the human ORFeome library. MIPSA identified previously known autoreactivities, and also detected undescribed neutralizing interferon lambda 3 (IFN-λ3) autoantibodies. At-risk individuals with anti- IFN-λ3 antibodies may benefit from interferon supplementation therapies, such as those currently undergoing clinical evaluation.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.

Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called 'Ligation in situ Hybridization' ('LISH'), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA-DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes ('LISH-seq'), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.

α-Synuclein-mediated inhibition of ATF6 processing into COPII vesicles disrupts UPR signaling in Parkinson's disease.

The unfolded protein response (UPR) monitors the folding environment within the endoplasmic reticulum (ER). Accumulation of misfolded proteins within the ER activates the UPR resulting in the execution of adaptive or non-adaptive signaling pathways. α-Synuclein (α-syn) whose accumulation and aggregation define the pathobiology of Parkinson's disease (PD) has been shown to inhibit ER-Golgi transit of COPII vesicles. ATF6, a protective branch of the UPR, is processed via COPII mediated ER-Golgi transit following its activation via ER stress. Using cellular PD models together with biochemical reconstitution assays, we showed that α-syn inhibited processing of ATF6 directly through physical interactions and indirectly through restricted incorporation into COPII vesicles. Impaired ATF6 signaling was accompanied by decreased ER-associated degradation (ERAD) function and increased pro-apoptotic signaling. The mechanism by which α-syn inhibits ATF6 signaling expands our understanding of the role ER stress and the UPR play in neurodegenerative diseases such as PD.

Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking.

Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.

Characterization of ricin toxin family members from Ricinus communis.

Ricin inhibits translation by removal of a specific adenine from 28S RNA. The Ricinus communis genome encodes seven full-length ricin family members. All encoded proteins have the ability of hydrolyzing adenine in 28S rRNA. As expected, these proteins also inhibited an in vitro transcription/translation system. These data show that the ricin gene family contains at least seven members that have the ability to inhibit translation and that may contribute to the toxicity of R. communis.

Genome-wide diversity and selective pressure in the human rhinovirus.

BACKGROUND: The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. RESULTS: To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. CONCLUSION: This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution.

On the mechanism of sensing unfolded protein in the endoplasmic reticulum.

Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-A crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by alpha-helices that are situated on a beta-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.