Skip to main content
. 2008 Dec 10;3(12):e3904. doi: 10.1371/journal.pone.0003904

Figure 2. Phenotype of PB2 mutants with altered NLS.

Figure 2

(A) Sequence of the PB2 NLS located close to the C-terminus. The position of the two regions of the bipartite NLS (NLS-1 and NLS-2) is indicated, as well as the mutations present in mutants 737, 738, KNRQ, 8/K and ΔNLS. (B) Representative intracellular localisations of wt and mutant PB2 proteins. Central optical sections are presented of HeLa cells mock-transfected (MOCK), transfected with wt PB2 (WT) or with each of the mutant PB2 proteins. Nuclei were stained with DAPI (blue) and PB2 was stained with anti-PB2 monoclonal antibody and goat anti-mouse IgG coupled with Alexa 488 (green). (C) Quantitative estimation of the localisation of wt or mutant PB2 proteins. Around 100 individual cells were scored as: N, exclusively nuclear; N>C, nuclei more stained than the cytoplasm; NC, equal nuclear and cytoplasmic staining or C, exclusively cytoplasmic staining. (D) Biological activity of recombinant RNPs with mutated PB2 proteins. Cultures of HEK293T cells were co-transfected with plasmids expressing PB1, PA, NP and either wt or mutant PB2, as well as a plasmid encoding CAT gene in negative polarity, flanked by the non-translated regions of influenza NS segment. Plasmid expressing PB2 was omitted as a negative control. At 20 hours post-transfection total cell extracts were prepared and CAT protein was determined by ELISA. The averages and standard deviations of 3–9 independent experiments are shown.