FIG 6.

MERS-CoV NS4b NLS and PDE catalytic mutants are attenuated in A549 cells and exhibit increased type III IFN expression. (A) Vero cells were infected in triplicate at an MOI of 1 with WT MERS-CoV, MERS-ΔNS4a, and MERS-ΔNS4ab. Supernatants were collected at indicated times postinfection and infectious virus quantified by plaque assay. (B) A549^DPP4 cells were infected in triplicate at an MOI of 1 or 0.1 with WT MERS-CoV, MERS-ΔNS4a, and MERS-ΔNS4ab, and replication was quantified as in panel A. Data are from one representative of three independent experiments and are displayed as means ± standard deviation (SD). (C) Statistical significance for mutant virus replication versus WT was determined by two-way ANOVA: *, P ≤ 0.05; **, P ≤ 0.01, ***, P ≤ 0.001; ****, P ≤ 0.0001. (D) A549^DPP4 cells were mock infected or infected in triplicate at an MOI of 5 with WT MERS-CoV, MERS-NS4b^NLS, and MERS-NS4b^H182R, and RNA was harvested at the indicated times postinfection. Gene expression over mock-infected cells was measured by RT-qPCR and calculated over mock-infected cells using the 2^−Δ(ΔCT) formula. Data are from one representative of three independent experiments and expressed as mean ± SEM. Statistical significance was determined by unpaired Student's t test: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (E) A549^mCEACAM-1 cells were mock treated or infected with WT MHV or MHV-NS2^H126R at an MOI of 5, and RNA was harvested at 6 and 12 h postinfection. IFNL1 expression was determined as in panel D. Data are from one representative experiment of three.