Similarities and differences in the conformational stability and reversibility of ORF8, an accessory protein of SARS-CoV-2, and its L84S variant

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has the characteristic accessory protein ORF8. Although clinical reports indicate that ORF8 variant strains (Δ382 and L84S variants) are less likely to cause severe illness, functional differences between wild-type and variant ORF8 are unknown. Furthermore, the physicochemical properties of the ORF8 protein have not been analyzed. In this study, the physicochemical properties of the wild-type ORF8 and its L84S variant were analyzed and compared. Using the tobacco BY-2 cell production system, which has been successfully used to produce the wild-type ORF8 protein with a single conformation, was used to successfully produce the ORF8 L84S variant protein at the same level as wild-type ORF8. The produced proteins were purified, and their temperature and pH dependencies were examined using nuclear magnetic resonance spectra. Our data suggested that the wild-type and L84S variant ORF8 structures are highly stable over a wide temperature range. Both proteins displayed an aggregated conformation at higher temperature that reverted when the temperature was decreased to room temperature. Moreover, ORF8 precipitated at acidic pH and this precipitation was reversed when the solution pH was shifted to neutral. Interestingly, the L84S variant exhibited greater solubility than wild-type ORF8 under acidic conditions. Thus, the finding indicated that conformational stability and reversibility of ORF8 are key properties related to function in oppressive environments.

Keywords: L84S variant; ORF8; Protein conformation; Protein stability; Reversibility; SARS-CoV-2.

Fig. 1

Production of ORF8 protein in tobacco BY-2 cells. (A) Schematic representation of tobamovirus (ToMV)-mediated chemically induced expression plasmids. XVE, transcriptional activator that responds to estrogen; SP, signal peptide of Arabidopsis chitinase; 8His, 8 × His-tag; ORF8; mature ORF8 coding sequence (CDS); ORF8(L84S), ORF8 L84S variant CDS; LBS, LexA binding site; P_G10-90, synthetic constitutive promoter; P_LexA-46, fusion promoter controlled by XVE; dMP, partial movement protein; SRz, ribozyme sequence from tobacco ringspot virus satellite RNA; 35ST, 35S terminator; RB, right border; and LB, left border. Hygr and Kanr denote the expression cassette of the hygromycin and kanamycin resistance genes, respectively. (B) Detection of wild-type ORF8 and its L84S variant via Coomassie brilliant blue staining without dithiothreitol. Culture medium represents samples from cells incucubated in culture medium supplemented with 17β-estradiol for 7 days. (C) Purified indicates ORF8s purified using a Ni²⁺ affinity column and size exclusion column. Arrowheads indicate ORF8. M, protein size marker. Numbers indicate molecular weights (kDa). (D) ¹H nuclear magnetic resonance (NMR) spectra of wild-type ORF8 (WT) and its L84S variant (L84S). The spectra were recorded on a 500 MHz-NMR spectrometer. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Methyl region of the ¹H nuclear magnetic resonance (NMR) spectra at various temperatures. Wild-type ORF8 (left) and the L84S variant (right) were analyzed. The spectra labeled with 30∗ were those recorded at 30 °C after heating.

Fig. 3

Methyl region of ¹H nuclear magnetic resonance (NMR) spectra at various pH values. Wild-type ORF8 (left) and the L84S variant (right) were analyzed. The peaks labeled with ∗ were resonated from impurity. The label “xN” means that the vertical axis was magnified N times as indicated by the height of an impurity peak at 0.08 ppm. The spectra were recorded at 25 °C on an 800 MHz-NMR spectrometer.

Fig. 4

Resolubilization of ORF8 proteins. Detection of wild-type ORF8 and its L84S variant via Coomassie brilliant blue staining. WT, wild-type ORF8; L84S, L84S variant. + and − indicate the presence and absence of 10 mM dithiothreitol, respectively. Arrowheads indicate ORF8. M, protein size marker. Numbers indicate molecular weights (kDa). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)