Journal: Journal of Virology
Article Title: Discontinuous template switching generates coronavirus subgenomic RNAs from the 3ʹ viral genome end by 5ʹ to 3ʹ transcription
doi: 10.1128/jvi.01438-25
Figure Lengend Snippet: Mutation of the mapped TRS B usage for synthesis of hCoV-OC43 NG-ns12.9 sgRNA activates the usage of a cryptic TRS B , 64 nt downstream. ( A ) Diagram showing where the silence mutations were introduced into the ns12.9 TRS B core (box) and/or adjacent regions to create five mutated NG-ns12.9 ds-circDNA products (MT-1 to MT-5). (B to D) Visualization and quantification of NG + HCT-8 cells after co-cultivation with HEK293T cells transfected with each CPER-derived hCoV-OC43 NG-ns12.9 ds-circDNA. Individual FL CPER products with or without TRS B mutations were directly co-transfected to HEK293T cells along with a hCoV-OC43 N protein expression vector pCOC42 and co-cultivated with HCT-8 cells for 7 days post transfection before FACS analysis. ( B ) NG + HCT-8 cells quantified by FACS analysis on D7 post-transfection. MK (mock infection), HCT-8 cells co-cultivated with untransfected HEK293T cells; WT, hCoV-OC43-ns12.9 with WT TRS B sequence; MT-1 to MT-5 (mutant-1 to mutant-5), hCoV-OC43-ns12.9 TRS B with mutated sequences of MT-1 to MT-5 shown in ( A ). Data were averaged from three independent experiments with one-tailed Student’s t -test. *, P < 0.05; ns, no statistical significance. ( C ) The median fluorescent intensity (MFI) profile is one representative FACS analysis of three experiments. ( D ) Representative microscopic images on D7 post-transfection showing NG + HCT-8 cells from WT or the indicated mutants. ( E ) RT-PCR on total RNA extracted from infected HCT-8 cells on D7 post co-cultivation. Gel electropherogram shows a 347 bp (band-1) product amplified from the hCoV-OC43 NG-ns12.9 sgRNA using a WT TRS B . A similar band of 347 bp (1*, 1$, and 1#) was also detected from the MT-1, MT-2, and MT-5 with indicated mutations in ( A ) and their correspondent sequences. The RT-PCR products from MT-1 to MT-5 mainly generated a 276 bp product (band-2) by using an alternative TRS B motif, 64 nt downstream of the WT ns12.9 TRS B . A nucleotide with a red box indicates the introduced mutation and with an underline indicates unexpected mutations. ( F ) Northern blot analysis of hCoV-OC43 NG-ns12.9 RNA from infected HCT-8 cells in co-cultivation with HEK293T cells transfected with the CPER-derived WT NG-ns12.9 TRS B ds-circDNA (lane 2) or a mutant NG-ns12.9 TRS B MT-1 to MT-5 ds-circDNA (lanes 3 to 7, respectively). Cells without transfection served as a mock infection (MK, lane 1). Total RNA extracted from the co-cultivated cells on D7 was analyzed by Northern blot using a 32 P-labeled probe antisense to the hCoV-OC43 N ORF. The bands correspondent to each sgRNA are labeled on the right. ( G ) Western Blot analysis of total protein extracted from infected HCT-8 cells in co-cultivation with HEK293T cells transfected with the CPER-derived WT NG-ns12.9 TRS B ds-circDNA (lane 1) or a mutant NG-ns12.9 TRS B (MT-1 to MT-5, lanes 2 to 7) ds-circDNA to detect the presence of spike protein with an hCoV-OC43 spike-specific polyclonal antibody. The intensity of each protein band was quantified and used to calculate the relative amount of spike protein (%). The human GAPDH was used as a loading control.
Article Snippet: Total cell lysates were resolved on a 4%–12% Bis–Tris NuPAGE gel (Thermo Fisher Scientific), transferred to a nitrocellulose membrane, and blotted with a rabbit anti-hCoV-OC43 Spike (S) protein antibody (E4U6P, #16435 Cell Signaling Technology) or mouse anti-GAPDH (D4C6R, #97166 Cell Signaling Technology) as a loading control.
Techniques: Mutagenesis, Transfection, Derivative Assay, Expressing, Plasmid Preparation, Infection, Sequencing, One-tailed Test, Reverse Transcription Polymerase Chain Reaction, Amplification, Generated, Northern Blot, Labeling, Western Blot, Control
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