Susceptibility of SARS COV-2 nucleocapsid and spike proteins to reactive oxygen species and role in inflammation.

Chemiluminescence was used to test the susceptibility of the SARS-CoV-2 N and S proteins to oxidation by reactive oxygen species (ROS) at pH 7.4 and pH 8.5. The Fenton's system generates various ROS (H2O2, OH, -OH, OOH). All proteins were found to significantly suppress oxidation (the viral proteins exhibited 25-60% effect compared to albumin). In the second system, H2O2 was used both as a strong oxidant and as a ROS. A similar effect was observed (30-70%); N protein approached the effect of albumin at physiological pH (∼45%). In the O2.--generation system, albumin was most effective in the suppression of generated radicals (75%, pH 7.4). The viral proteins were more susceptible to oxidation (inhibition effect no more than 20%, compared to albumin). The standard antioxidant assay confirmed the strong antioxidant capacity of both viral proteins (1.5-1.7 fold higher than albumin). These results demonstrate the effective and significant inhibition of ROS-induced oxidation by the proteins. Obviously, the viral proteins could not be involved in the oxidative stress reactions during the course of the infection. They even suppress the metabolites involved in its progression. These results can be explained by their structure. Probably, an evolutionary self-defense mechanism of the virus has been developed.

Capabilities of Double-Resonance LPG and SPR Methods for Hypersensitive Detection of SARS-CoV-2 Structural Proteins: A Comparative Study.

The danger of the emergence of new viral diseases and their rapid spread demands apparatuses for continuous rapid monitoring in real time. This requires the creation of new bioanalytical methods that overcome the shortcomings of existing ones and are applicable for point-of-care diagnostics. For this purpose, a variety of biosensors have been developed and tested in proof-of-concept studies, but none of them have been introduced for commercial use so far. Given the importance of the problem, in this study, long-period grating (LPG) and surface plasmon resonance (SPR) biosensors, based on antibody detection, were examined, and their capabilities for SARS-CoV-2 structural proteins detection were established. Supersensitive detections of structural proteins in the order of several femtomoles were achieved by the LPG method, while the SPR method demonstrated a sensitivity of about one hundred femtomoles. The studied biosensors are compatible in sensitivity with ELISA and rapid antigen tests but, in contrast, they are quantitative, which makes them applicable for acute SARS-CoV-2 infection detection, especially during the early stages of viral replication.

Binding of SARS-CoV-2 Structural Proteins to Hemoglobin and Myoglobin Studied by SPR and DR LPG.

One of the first clinical observations related to COVID-19 identified hematological dysfunctions. These were explained by theoretical modeling, which predicted that motifs from SARS-CoV-2 structural proteins could bind to porphyrin. At present, there is very little experimental data that could provide reliable information about possible interactions. The surface plasmon resonance (SPR) method and double resonance long period grating (DR LPG) were used to identify the binding of S/N protein and the receptor bind domain (RBD) to hemoglobin (Hb) and myoglobin (Mb). SPR transducers were functionalized with Hb and Mb, while LPG transducers, were only with Hb. Ligands were deposited by the matrix-assisted laser evaporation (MAPLE) method, which guarantees maximum interaction specificity. The experiments carried out showed S/N protein binding to Hb and Mb and RBD binding to Hb. Apart from that, they demonstrated that chemically-inactivated virus-like particles (VLPs) interact with Hb. The binding activity of S/N- and RBD proteins was assessed. It was found that protein binding fully inhibited heme functionality. The registered N protein binding to Hb/Mb is the first experimental fact that supports theoretical predictions. This fact suggests another function of this protein, not only binding RNA. The lower RBD binding activity reveals that other functional groups of S protein participate in the interaction. The high-affinity binding of these proteins to Hb provides an excellent opportunity for assessing the effectiveness of inhibitors targeting S/N proteins.

SPR-Based Kinetic Analysis of the Early Stages of Infection in Cells Infected with Human Coronavirus and Treated with Hydroxychloroquine.

Cell-based assays are a valuable tool for examination of virus-host cell interactions and drug discovery processes, allowing for a more physiological setting compared to biochemical assays. Despite the fact that cell-based SPR assays are label-free and thus provide all the associated benefits, they have never been used to study viral growth kinetics and to predict drug antiviral response in cells. In this study, we prove the concept that the cell-based SPR assay can be applied in the kinetic analysis of the early stages of viral infection of cells and the antiviral drug activity in the infected cells. For this purpose, cells immobilized on the SPR slides were infected with human coronavirus HCov-229E and treated with hydroxychloroquine. The SPR response was measured at different time intervals within the early stages of infection. Methyl Thiazolyl Tetrazolium (MTT) assay was used to provide the reference data. We found that the results of the SPR and MTT assays were consistent, and SPR is a reliable tool in investigating virus-host cell interaction and the mechanism of action of viral inhibitors. SPR assay was more sensitive and accurate in the first hours of infection within the first replication cycle, whereas the MTT assay was not so effective. After the second replication cycle, noise was generated by the destruction of the cell layer and by the remnants of dead cells, and masks useful SPR signals.