Generation of MERS-CoV-Δ4a and Δ4b deletion mutants.

<p>(A) Schematic representation of MERS-CoV WT genome. Essential viral genes (ORF1a, ORF1b, S, E, M, N) and accessory genes (3, 4a, 4b and 5) are indicated. L, leader sequence; A_n, poly(A) tail; CS, conserved sequence included in transcription-regulating sequences. Genes 4a and 4b were deleted from the MERS-CoV infectious cDNA clone by PCR-directed mutagenesis as described in Materials and Methods. Deleted regions within 4a and 4b genes are illustrated as light grey boxes. The size of both deleted and remaining regions in 4a and 4b genes is indicated by arrows. The patterned boxes in Δ4a and Δ4b mutants indicate frameshift mutations in 4a and 4b sequence caused by the deletions. (B) Growth kinetics of Δ4a and Δ4b deletion mutants in Huh-7 cells at the indicated MOIs. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. (C) NF-κB-dependent cytokine response of Huh-7 cells either mock-infected or infected with WT, Δ4ab, Δ4a or Δ4b mutants (MOI = 1 PFU/cell). At 24 hpi, mRNA levels of IL-6, IL-8 and TNF-α were quantified by RT-qPCR and compared to those in WT-infected cells, using the ΔΔCt method for calculation and HMBS as a reference endogenous gene. Shown are means with standard deviations, which were analyzed using an unpaired t-test against the wild-type (**, p<0.01; ***, p<0.001).