Fig. 4

Synthesis of noncanonical coronavirus transcripts is regulated under different infection conditions. A Diagram depicting the probes used for Northern blotting in Figures (B–D). B Left panel: Diagram depicting the preparation of BCoV inoculum from different passages and origins. For the BCoV inoculum from different passages, HRT-18 cells were infected with 0.1 MOI of BCoV inoculum (designated viral passage 0 BCoV; VP0 BCoV). After 48 h of infection, total cellular RNA (VP0 RNA) was harvested, and the supernatant (designated VP1 BCoV) was collected to infect another batch of fresh HRT-18 cells. For BCoV inoculum from different cell origins, HEK-293T cells were infected with 0.1 MOI of BCoV inoculum (VP0 BCoV). After 48 h of infection, the supernatant (designated VP1 HBCoV) was collected to infect another batch of HRT-18 cells at an MOI of 0.1. Right panel: Detection of noncanonical transcripts from HRT-18 cells infected with BCoV from different passages or origins by Northern blotting. The RNA species representing noncanonical transcripts between passages (Lanes 2 and 4) and origins (Lanes 4 and 6) are denoted by brown bars. C Detection of noncanonical transcripts from the same HRT-18 cells infected with different MOIs (0.1 and 10) of BCoV by Northern blotting. The RNA species representing noncanonical transcripts between different MOIs (Lanes 4 and 5) at 48 hpi are denoted by blue bars. D Comparison of the species and amounts of noncanonical transcripts between passages from fresh HRT-18 cells infected with the BCoV-p95 isolate. Left panel: Diagram depicting different passages of RNA collected from fresh HRT-18 cells infected with BCoV-p95 inoculum (VP0 BCoV-p95) and supernatant from different passages of BCoV-p95 (VP1-VP6 BCoV-p95). Right panel: Detection of noncanonical transcripts from the aforementioned total cellular RNA by Northern blotting. The RNA species representing noncanonical transcripts are denoted by orange and blue bars. kb, kilobase; m, mock-infected cells; hpi, hours postinfection; 28S, 28S rRNA