Highlights d A quick PCR-based reverse genetics system is established for SARS-CoV-2 d SARS-CoV-2 recombinants harboring reporter genes or mutations can be generated

No abstract

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.Mutations in Cu,Zn-superoxide dismutase (SOD1) 2 are linked to familial forms of amyotrophic lateral sclerosis (fALS) (1). A major pathological change observed in SOD1-related fALS is the abnormal accumulation of misfolded mutant SOD1 proteins in affected motor neurons (2). Actually, many in vivo as well as in vitro studies have supported that pathogenic mutations facilitate the misfolding of SOD1 proteins (3); however, the molecular mechanism triggering the misfolding of SOD1 remains controversial.SOD1 is known as one of the most stable proteins to the extent that its melting temperature (T m ) is Ͼ90°C (4); therefore, a misfolding event appears quite unlikely for SOD1. Nonetheless, SOD1 was found to have acquired such high stability through several post-translational processes including copper and zinc binding and disulfide bond formation (Fig. 1A). Actually, disulfide-reduced apoSOD1 exhibits significantly decreased stability (T m ϳ 42°C) and is susceptible to unfolding/ misfolding at physiological temperatures (5, 6). Intracellular deregulation of metal binding and/or disulfide formation will, hence, be a key event triggering the misfolding of SOD1.Notably, many pathogenic mutations are found to disturb the post-translational control of SOD1 maturation (7, 8) and thereby increase intracellular fractions of the apo-(9) and/or disulfide-reduced state (10). Only when both metal ions and disulfide bond are absent, SOD1 forms fibrillar aggregates (11). Given that SOD1 fibrillation is a pathological hallmark in SOD1-related fALS patients (12) as well as model mice (13), the most immature form of SOD1 will provide a clue to understand the molecular pathomechanism of this devastating disease. In a number of previous studies, the roles of metal binding and disulfide formation in the misfolding of SOD1 have been suggested by a variety of experimental methods (6, 8, 11, 14 -19); however, conformational information on SOD1 in solution lacking both metal ions and the disulfi...

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.