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. 2021 Apr 26;95(10):e00140-21. doi: 10.1128/JVI.00140-21

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FIG 1 — Mutation at putative S protein cleavage sites influences the production of HCoV-OC43 infectious virus. (A) Representation of the full-length HCoV-OC43 genome found within the pBAC-OC43^FL infectious clone with a schematic representation of the HCoV-OC43 S gene with the two cleavage sites: S1/S2 and putative S2′ cleavage site (black arrows). The S protein is composed of two subunits, S1, containing the receptor binding domain (RBD) and hypervariable region (HVR), and S2, containing the fusion subunit. L, linker region between S1/S2 and S2′ sites; FP, putative fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane domain; E, endodomain; ORF, open reading frame. Not drawn to scale. Data in table represent corresponding cleavage sequences of the different viruses, including reference pBAC-OC43^FL. (B) Infectious virus production evaluated after transfection of BHK-21 cells (BHK-P0) and two rounds of amplification in HRT-18 cells (HRT-18 P1 and P2). (C) RT-PCR analysis for the presence of viral RNA after transfection of BHK21. Lane 1 is the negative control (ctrl) (transfected with wild-type pBAC-OC43^FL without reverse transcription step), lane 2 is wild-type pBAC-OC43^FL, lane 3 is pBAC-OC43/S_G758R, lane 4 is clone 1 of pBAC-OC43/S_R903A, lane 5 is clone 2 of pBAC-OC43/S_R903A, lane 6 is clone 1 of pBAC-OC43/S_G758R-R903A, lane 7 is clone 2 of pBAC-OC43/S_G758R-R903A, and lane 8 is clone 3 of pBAC-OC43/S_G758R-R903A. MW, molecular weight standards. (D) RT-PCR analysis for the presence of viral RNA in HRT-18 cells. Lane 1 is the negative ctrl (RNA extracted from OC43-infected cells without reverse transcription step), and lane 2 is the positive ctrl (RNA extracted from OC43-infected cells). Other lanes show RT-PCR performed on RNA extracted after two rounds of amplification (infection) in HRT-18 cells (HRT-18 P2): lane 3 comes from clone 1 of pBAC-OC43/S_G758R-R903A, lane 4 comes from clone 2 of pBAC-OC43/S_G758R-R903A, lane 5 comes from clone 3 of pBAC-OC43/S_G758R-R903A, lane 6 comes from clone 1 of pBAC-OC43/S_R903A, lane 7 comes from clone 2 of pBAC-OC43/S_R903A, lane 8 comes from pBAC-OC43/S_G758R, and lane 9 comes from wild-type pBAC-OC43^FL.