The coronavirus disease-2019 (COVID-19), caused by the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (Harrison et al., 2020), belongs to the order Nidovirales (Gulyaeva & Gorbalenya, 2021), which includes viruses with a single-stranded, positive-sense RNA with large genome ranging in size from 26 to 32 kb (Gorbalenya et al., 2020; Gorbalenya et al., 2006). SARS-CoV-2 has a genome of ~ 30 kb. (Malone et al., 2022). This large genome is annotated to possess 14 open reading frames (ORFs) encoding 9890 amino acids, making 27 proteins (Gorbalenya et al., 2006; Wu et al., 2020). SARS-CoV-2 comprises four main structural proteins, including envelope, membrane, nucleocapsid, and spike proteins (Huang et al., 2020). The fragment of spike protein, from amino acid 319 to 541, is known as the receptor-binding domain (RBD) (Shin et al., 2021), which plays a central role in the disease process by binding to the angiotensin converting enzyme-2 (ACE2) receptor, allowing the virus to enter the host cell and initiate infection (Li et al., 2003).
According to the World Health Organization (WHO), 773,819,856 confirmed cases have been infected with COVID-19, resulting in 7,010,568 deaths (WHO Coronavirus (COVID-19) Dashboard, 2023). Alongside the mortality caused by COVID-19, the long-term effects of this disease and the enduring effects of vaccines are still under discussion and investigation (Byambasuren et al., 2023). In the meantime, there are reports of COVID-19 patients suffering from an increase in the level of Alzheimer’s disease (AD) progression (Chen et al., 2022; Patel et al., 2022) as well as an elevation in the level of β-amyloid (Aβ) in the blood (Hsu et al., 2021), interacting directly with fibrin and fibrinogen to cause blood hypercoagulation (Grobbelaar et al., 2021) and diabetes, which is linked to islet amyloid polypeptide (IAPP) (Young et al., 2015). All of these reports are associated with amyloidotic fibrillar aggregates. One important question is which SARS-CoV-2 structural proteins can form amyloid, as this knowledge is vital for understanding the pathogenesis of the disease, including long COVID-19 (Leung et al., 2023).
Amyloid fibril formation has a connection with the increased content of β-structure in a protein structure, resulting in its fibrillar aggregation (Jimenez et al., 1999). Remarkably, the SARS-CoV-2 proteins comprise various amyloid-forming sequences (Milton, 2023; Tayeb-Fligelman et al., 2023); among them, the spike protein contains RBD, with a secondary β-structure content of 34.1% (Brindha & Kuroda, 2022; Nystrom & Hammarstrom, 2022). RBD is a vaccine candidate and a target for drug development (Fitzgerald et al., 2021); however, the amyloidogenicity of RBD is still unclear, and the main related question is the possibility of the SARS-CoV-2 RBD amyloidogenic characteristics and its difference among various SARS-CoV-2 strains. Due to the rapid mutation of SARS-CoV-2, certain species presently do not exist, and only Omicron subvariants have become prevalent worldwide (Farahat et al., 2022; Parums, 2023). Consequently, our experiments were conducted with a particular emphasis on Omicron RBD, aiming to investigate the potential of RBD in the formation of amyloid fibrils and to identify the impact of SUMO fusion Tag on this phenomenon. To elucidate the underlying reasons for the variations in amyloid formation potential within the RBD across different strains of SARS-CoV-2, we compared the amyloidogenic potential of RBD among two significant variants of SARS-CoV-2 (Delta Plus and Omicron strains) using the FoldAmyloid algorithm (Garbuzynskiy et al., 2010), an in silico method of predicting amyloidogenic regions in a protein sequence. As fusion tags can provide control over the aggregation of an amyloidogenic protein (Kronqvist et al., 2022), the impact of the small ubiquitin-like modifier (SUMO) tag, which can enhance protein solubility due to its external hydrophilic surface and inner hydrophobic core (Butt et al., 2005), was examined on the Thioflavin T (ThT) fluorescence intensity of RBD amyloid-like fibrils formation. The findings will be presented based on the analysis of purified RBD of Delta Plus and RBD of Omicron variant with and without SUMO tag, isolated from Escherichia coli (Fig. 1) using ThT kinetics assay, fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM).