Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19

Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion ▿

Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the “pH sensor” that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer

The Arabidopsis COP9 Signalosome Subunit 7 Is a Model PCI Domain Protein with Subdomains Involved in COP9 Signalosome Assembly[W]

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 Å crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits

Selective Pressure Causes an RNA Virus to Trade Reproductive Fitness for Increased Structural and Thermal Stability of a Viral Enzyme

<div><p>The modulation of fitness by single mutational substitutions during environmental change is the most fundamental consequence of natural selection. The antagonistic tradeoffs of pleiotropic mutations that can be selected under changing environments therefore lie at the foundation of evolutionary biology. However, the molecular basis of fitness tradeoffs is rarely determined in terms of how these pleiotropic mutations affect protein structure. Here we use an interdisciplinary approach to study how antagonistic pleiotropy and protein function dictate a fitness tradeoff. We challenged populations of an RNA virus, bacteriophage Φ6, to evolve in a novel temperature environment where heat shock imposed extreme virus mortality. A single amino acid substitution in the viral lysin protein P5 (V207F) favored improved stability, and hence survival of challenged viruses, despite a concomitant tradeoff that decreased viral reproduction. This mutation increased the thermostability of P5. Crystal structures of wild-type, mutant, and ligand-bound P5 reveal the molecular basis of this thermostabilization—the Phe207 side chain fills a hydrophobic cavity that is unoccupied in the wild-type—and identify P5 as a lytic transglycosylase. The mutation did not reduce the enzymatic activity of P5, suggesting that the reproduction tradeoff stems from other factors such as inefficient capsid assembly or disassembly. Our study demonstrates how combining experimental evolution, biochemistry, and structural biology can identify the mechanisms that drive the antagonistic pleiotropic phenotypes of an individual point mutation in the classic evolutionary tug-of-war between survival and reproduction.</p> </div

Thermal and proteolytic stabilities of P5^wt and P5^V207F.

<p>A) Thermal melting of P5^wt and P5^V207F measured by circular dichroism (CD) spectrometry at 220 nm. P5^wt and P5^V207F began to unfold cooperatively at 50–55°C (left panel). Melting temperatures were calculated from the second derivative of the CD melting curves (right panel). The V207F mutation increases the melting temperature of P5 by 7.6°C. B) Limited proteolysis of P5ΔV8^wt and P5ΔV8^V207F with V8 (Glu-C) protease monitored by SDS-PAGE. Some minor proteolytic products (marked by asterisks) are visible in P5ΔV8^wt. C) Thermal melting curves of P5ΔV8^wt and P5ΔV8^V207F, with melting temperatures calculated as in (A). D) Differential Scanning Calorimetry (DSC) of P5 and P5ΔV8 proteins. Peaks indicate a 6°C difference between the melting points of wildtype and mutant P5.</p

The structure of P5 bound to a glycan suggests lytic transglycosylase activity.

<p>A) Overall structure of P5ΔV8^wt bound to chitotetraose. The 2F_o - F_c electron density map for the ligand is shown contoured at 1 σ. Residues 199–220 are disordered. Helix α7 and the following linker are in slightly different positions than in the unliganded structure (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003102#pgen.1003102.s003" target="_blank">Figure S3</a>). The four NAG residues of chitotetraose bind to subsites A–D. B) Close-up of the active site. A water molecule is observed between Glu95 and the NAG in subsite D, supporting a lytic transglycosylase (LT) activity, with Glu95 as the catalytic acid/base. C) Superposition of ligand-bound P5ΔV8^wt (yellow) and apo-P5ΔV8^wt (orange) onto the structure of the <i>E. coli</i> slt70 LT (brown) containing a glycan product in subsites E and F (PDB 1QTD). The geometry and electrostatics of the P5 substrate-binding surface (D) are similar to those of slt70 (E). P5 residues displaced by the chitotetraose ligand are shown in grey with a semi-transparent surface.</p