Figure 2

The C-terminal end of the NS1 protein in H3N2 type influenza A viruses encode for a functional NoLS. (A) The N- and C-terminal fragments of the NS1 genes, encoding for amino acids 1–73 and 157–237 in A/Udorn/72, amino acids 1–73 and 157–230 in A/WSN/33 and amino acids 203–230 in A/Brevig Mission/1/18, were inserted into the GFP expression vector pCMX-SAH/Y145F to express GFP-NS1 fusion proteins. Mutations in the C-terminal NLS2/NoLS of A/Udorn/72 virus NS1 protein were performed as described in Materials and Methods. Red boxes indicate the positions of N-terminal NLS1 and C-terminal NLS2/NoLS in NS1 protein. (B) HuH7 cells were transiently transfected with GFP, GFP-NS1 fusion and GFP-NS1 fusion mutant (K219A,R220A + R231A,R232A) A/Udorn/72 gene constructs, as indicated in the figure, for 48 h. The intensity of nucleolar localization was scored by immunofluorescence microscopy as no nucleolar staining (−) or weak (+), moderate (++) or strong (+++) nucleolar staining. Critical basic amino acids involved in nuclear/nucleolar targeting are marked in boldface and underlined. Mutated amino acids (K219A,R220A + R231A,R232A) are marked in red. (C) HuH7 cells were transiently transfected with GFP and the GFP-deletion gene constructs of NS1 A/WSN/33 and NS1 A/Brevig Mission/1/18 for 48 h as indicated in the figure. Critical and mutated amino acids are marked as above. Bars, 5 μm.