Figure 4

Identification of the NLS motifs (BiNLS1 and NLS2) in VP2. (A) Substitutive mutagenesis was used to create three constructs, VP2 150-152 (RKK to AAA), VP2 136-138 (KRK to AAA), as well as double and triple mutations, within the BiNLS1 region of VP2 (also saw in Table 1). The subcellular localization of these mutants (green) were examined by fluorescent microscopy in HeLa and CHO cells. (B) To further identify the functional NLS motif, K133 and R134 were replaced by alanine. The mutants (VP2 136-138A/134A, VP2 136-138A/133A and VP2 136-138A/133A/134A) were then expressed and examined by fluorescent microscopy (green). All cells were fixed and stained with DAPI (blue). (C) Identification of NLS2 in VP2. The point mutations VP2 133A, VP2 134A and VP2 133A/134A were transfected into HeLa cells. After 48 h post-transfection, the distributions of GFPs (green) were monitored by fluorescence microscopy. The cells were fixed and stained with DAPI (blue)