FIGURE 4.

Mutation of residues K214 and R260 to alanine (KRm) abolishes nucleolar targeting of P3. (A) Schematic of the P3 isoform of RABV P protein; P3 is expressed by translation initiation from the codon corresponding to Met‐53 of the full‐length P protein. The N‐terminal region (NTR; residues 53–182) containing the N‐terminal NLS (N‐NLS), and the globular CTD, the C‐terminal NLS and NES (C‐NLS and C‐NES, respectively) are indicated. K214 and R260 form part of the C‐NLS and are substituted for Ala in P3 KRm. Residue numbering corresponds to the full‐length P protein sequence. (B, C) HeLa cells were transfected to express the indicated proteins and treated with or without LMB (3 h) before imaging by live‐cell CLSM at 16–20 h post‐transfection (p.t), as previously. ²⁰ Yellow arrowheads indicate nucleoli. Magnified sections (zoom) of untreated cells are highlighted in yellow boxes. (C) Images such as those B (+ LMB) were used to determine the Fnu/n for individual cells (mean Fnu/n ± SEM, n ≥ 27 cells) as previously. ²⁷ , ⁴⁶ Statistical analysis used Student's t‐test; ****p < 0.0001. (D) HeLa cells transfected to express the indicated proteins were fixed and immunolabelled for Treacle using AlexaFluor‐647‐conjugated secondary antibodies (red fluorescence). Images are representative of ≥5 fields of view. Yellow boxes highlight regions magnified in the Zoom panels. White arrowhead in HeV M K258A panels highlights regions depleted of GFP fluorescence within FC‐DFC. Nuclei/DNA was labelled using Hoechst 33342 dye.