FIG 6.

ns12.9 accessory protein does not affect the early steps of HCoV-OC43 infection and protein expression. (A) Knockout of ns12.9 gene does not impair the entry process. RD cells were infected with HCoV-OC43-WT or HCoV-OC43-Δns12.9 at an MOI of 1 and fixed at 0.5 and 1.5 hpi. Cells were stained with an OC43-N mouse monoclonal antibody, and the Alexa Fluor 488-conjugated goat anti-mouse antibody was used as the secondary antibody. The stained cells were analyzed by flow cytometry. The results are representative of three independent experiments. (B) Knockout of ns12.9 gene does not impair viral RNA replication. Cells were infected with HCoV-OC43-WT or HCoV-OC43-Δns12.9 at an MOI of 1, and total RNA was extracted at 2, 4, 6, and 8 hpi. Viral RNA levels were analyzed by qRT-PCR with the OC43-RT primers. (C) Knockout of ns12.9 gene does not impair sgmRNA synthesis. Total RNA was extracted from the infected cells at 8 hpi, and HCoV-OC43 sgmRNA levels were analyzed by qRT-PCR using the specific primers presented in Table 1. Data represent the means ± SD and were generated from three independent experiments. Statistical significance: **, P < 0.01. (D) Knockout of ns12.9 gene does not impair OC43-N protein expression. Cell lysates were collected at 12, 18, and 24 hpi, and the expression levels of OC43-N protein were determined by Western blotting. The bands were analyzed and the fold changes of N protein at the indicated times were calculated considering the wild-type infected cells as 1 with actin as an internal control.