FIGURE 2.

Generation, verification, and rescue of MERS-CoV-Δ8b. (A and B) HEK293 cells were transfected with either empty vector or ORF8b-V5 expression construct. Transfected cells were harvested 40 h posttransfection for Western blot analysis with anti-V5 (α-V5) or anti-ORF8b (α-ORF8b) Abs. (C) HEK293 cells were transfected with either empty vector or ORF8b-V5 plasmid. Transfected cells were harvested 40 h posttransfection for immunoprecipitation (IP) with anti-ORF8b Ab. (D) Huh-7 cells were either mock infected or infected with MERS-CoV at an MOI of 0.01. Infected cells were harvested 24 hpi for Western blot analysis with anti-ORF8b Ab. The band indicated by the arrow corresponds to the size of ORF8b. (E) Schematic diagram showing pBeloBAC11-based molecular clone harboring the complete genome of MERS-CoV strain EMC-2012. Essential (ORF1a, ORF1b, S, E, M, and N) and accessory (3, 4a, 4b, 5, and 8b) genes are indicated. The ORF8b region was zoomed in. Overlapping region of N and ORF8b genes of WT and ORF8b-deficient (Δ8b) viruses was compared side by side, highlighting (red boxes) the point mutations affecting the nucleotide and amino acid sequences. The two premature stop codons introduced to ORF8b sequence are indicated by asterisks (*). The amino acid sequence of N protein remains unchanged. (F) Sequencing result of Δ8b clones. The desired point mutations indicated by red boxes were found in two independent clones. Because no difference in sequence was observed between these two clones, results derived from only one clone (clone 3) were presented hereafter. (G) Huh7 cells were either mock infected or infected with WT or Δ8b MERS-CoV for 48 h before harvested for Western blot analysis with anti-ORF8b. Virus production was confirmed by detecting N protein expression using anti-N Ab.