Abstract 1316 Identifying the Oligomerization State of the Transmembrane Domain of the ORF7b Accessory Protein Found in SARS-CoV-2 & Exploring Potential Means of Reducing Viral Pathogenicity

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal disease for the Coronavirus Disease-19 (COVID-19) has to date resulted in 771 confirmed cases and 6.9 million associated deaths. Vaccines are available to the public and focus on the disrupting the attachment of the Spike (S) protein to Angiotensin-converting enzyme 2 (ACE2) receptors. However, it is imperative to further investigate transmembrane domains of SARS-CoV-2 accessory proteins to explore the development of alternative methods to inhibit proliferation of the virus. The eleven SARS-CoV-2 accessory proteins are essential for cellular function, but their role in viral proliferation is still widely unknown. The functions of Open Reading Frame 7b (ORF7b) protein of SARS-CoV-2 are to induce apoptosis and promote Interferon-B (IFN-B), Interleukon-6 (IL-6), Tumor Necrosis-A (TNF-a), and to activate type-I IFN signaling through Interferon regulatory factor 3, IRF3, phosphorylation. The oligomerization state of the OR7b accessory protein has been observed to be a homotetramer in detergent. However, the oligomerization state of the transmembrane domain itself is not widely researched. The transmembrane domain of accessory protein ORF7b of SAR-CoV-2 was synthesized to identify its oligomerization state using a fluorescence assay. By determining how ORF7b folds, the synthesis of an inhibitor can be explored. The 21-residue long ORF7b accessory protein with an addition of four lysines at the C-terminus, to total 25 residues, was synthesized using an automated peptide synthesizer. Three variants of the peptide were synthesized: one without fluorophore, one with donor fluorophore, and one with quencher fluorophore. The peptides with fluorophores were chemically linked to the N-terminus of the peptide. In each case, the peptide is cleaved from the resin then purified and analyzed using Reverse-Phase High Performance Liquid Chromatography (HPLC) and Liquid Chromatography Mass Spectrometry (LCMS). To determine the oligomerization state of the transmembrane domain, all three variants of peptide are being used in a fluorescence-based assay. Ultimately, the oligomerization state of the transmembrane domain of ORF7b will provide crucial knowledge to allow the development of proof-of-concept inhibitors to hinder viral infectivity. Research was funded by the Hampden-Syndey College Department of Undergraduate Research.