Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2.

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2' cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Interaction of HSPA5 (Grp78, BIP) with negatively charged phospholipid membranes via oligomerization involving the N-terminal end domain.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.