Generation of MERS-CoV-4b-NLS mutants.

<p>(A) Schematic representation of 4a and 4b overlapping sequences in MERS-CoV WT (top) and 4b-NLS mutants (bottom). Nuclear localization signals in 4b protein (NLS-S1 and NLS-S2), consisting of positively charged amino acids lysine (K) and arginine (R) are indicated. The 189 nt-sequence duplicated in 4b-NLS mutants is indicated by the shadowed area and the arrows. DUP, mutant including WT duplicated 4a-4b sequences, used as a control; DUP-mNLS-S1, mutant with 4b NLS Site 1 mutated to alanine; DUP-mNLS-S2, mutant with Site 2 and two close Lys residues mutated to alanine, as described in Materials and Methods. The light blue boxes in DUP, DUP-mNLS-S1 and DUP-mNLS-S2 mutants indicate duplicated 4a sequences. PacI and NheI restriction sites used for the assembly of infectious cDNA clones and their genomic positions (first nucleotide of the recognition sequence) are indicated. (B) Analysis by confocal microscopy of 4b subcellular localization in Huh-7 cells either mock-infected or infected with MERS-CoV-4b-NLS mutants (MOI = 0.1 PFU/cell, 24 hpi). 4b protein (green) and dsRNA (red) were detected with specific antibodies, while nuclei (blue) were stained with DAPI. (C) Growth kinetics of MERS-CoV-4b-NLS mutants in Huh-7 and Calu-3 cells at an MOI of 0.1. Supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. Error bars represent SD.