Synthesis and Analysis of the SARS-CoV-2 ORF7a Accessory Protein Transmembrane Domain and Determination of the Oligomerization State

The ongoing SARS-CoV-2 pandemic continues to sicken millions worldwide and fundamentally change the way people interact with each other. In order to better characterize the SARS-CoV-2 virus and potentially develop methods of inhibition for further spread of the disease, this research project focused on synthesizing and characterizing the trans-membrane region of the accessory protein ORF7a. ORF7a has been implicated in proper viral assembly, leading to the idea that inhibition of this protein could prevent viral copies from being produced and halt the spread of the virus. The goal of this project was to determine the oligomerization state of the protein through a fluorescence assay in order to better understand the quaternary structure of the ORF7a complex and how it folds. The fluorescence assay is performed using three different samples of the synthesized peptide: one labeled with a TAMRA fluorophore, one labeled with a NBD fluorophore, and the last is unlabeled. After determining the oligomerization state of the protein, potential inhibitors could be synthesized and tested for their efficacy at inhibiting the function of the protein. Further applications of these inhibitors on other viruses can be explored due to the highly conserved nature of transmembrane domains across multiple viral families. Synthesis of the protein was done using a Solid Phase Peptide Synthesis (SPPS) technique and multiple batches of all three samples of peptide have been generated. Characterization and purification were done using High Performance Liquid Chromatography (HPLC) as well as Liquid Chromatography Mass Spectrometry (LCMS). Current research focuses on the purification and quantification of purified ORF7a oligopeptide for implementation of the fluorescence assay. -Hampden-Sydney College Office of Undergraduate Research.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Non-RBD binding antibody neutralize SARSCoV- 2

Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.

Metagenomic Discovery of a Novel Sarbecovirus in the United Kingdom at the Western Extreme of the Range of Rhinolophidae

Purpose: The origin of the current COVID-19 pandemic is unknown but horseshoe bats, of the family Rhinolophidae, are natural hosts to a suite of sarbecoviruses. Global surveillance is key to monitoring potentially pathogenic viral strains and improving the capacity for surveillance across Europe will bolster our understanding of viral populations within zoonotic reservoirs. Methods & Materials: Faecal samples were collected from Lesser horseshoe bats (Rhinolophus hipposideros) in the UK during annual population monitoring surveys, stored in RNAlater and frozen prior to genomic analysis. For metagenomic analysis, the Sequence-independent Single-Primer Amplification (SISPA) method was employed and sequencing completed using Illumina Nextera and the Oxford Nanopore GridION platforms. Results: A De novo hybrid assembly utilising shorter Illumina reads with longer Nanopore reads acting as a scaffold, generated a 29kb contig named RhGB01. Mapping raw reads against RhGB01 demonstrated a combined depth of 50x across the genome. Sequence alignment exhibits genomic organisation comparable to other sarbecoviruses isolated from animal and human hosts. Within the receptor binding domain, but excluding the receptor binding motif, RhGB01 has 77% and 81% amino acid homology compared to SARS-CoV-2 and SARS respectively. Maximum likelihood phylogenies inferred from the nucleotide sequence of RNA dependent RNA polymerase, spike glycoprotein and entire coding sequence exhibit clustering with the only other fully sequenced zoonotic Sarbecovirus from Europe which was isolated from Rhinolophus blasii. The structure of the receptor binding domain of RhGB01 was predicted by homology modelling using a crystal structure of the receptor binding domain of SARS-CoV as a template. This model was selected with a Global Model Quality Estimate (GMQE) > 0.7 and Quaternary Structure Quality Estimate (QMEAN) of -2.18. Structural comparisons between the predicted receptor binding domain of RhGB01 and SARS-CoV-2 highlight structurally different regions which house hACE2 contact residues. Conclusion: Phylogenetic inference and structural modelling suggest an absence of pathogenic potential for RhGB01. However, the discovery of a novel Sarbecovirus at the western limit of Lesser horseshoe bats demonstrates their presence throughout the entire horseshoe bat distribution and indicates the need for viral surveillance systems in Western Europe.

Research advances and prospects of legume lectins

Lectins are widely distributed proteins having ability of binding selectively and reversibly with carbohydrates moieties and glycoconjugates. Although lectins have been reported from different biological sources, the legume lectins are the best-characterized family of plant lectins. Legume lectins are a large family of homologous proteins with considerable similarity in amino acid sequence and their tertiary structures. Despite having strong sequence conservation, these lectins show remarkable variability in carbohydrate specificity and quaternary structures. The ability of legume lectins in recognizing glycans and glycoconjugates on cells and other intracellular structures make them a valuable research tool in glycomic research. Due to variability in binding with glycans, glycoconjugates and multiple biological functions, legume lectins are the subject of intense research for their diverse application in different fields such as glycobiology, biomedical research and crop improvement. The present review specially focuses on structural and functional characteristics of legume lectins along with their potential areas of application.