Fig 1.
Domain structure of RABV P-protein.
P-protein is shown schematically with key domains/sequences indicated; residue positions are indicated by italicized numbering. The RABV P gene encodes full length P1 (residues 1–297) and N-terminally truncated isoforms P2-P5 (expressed via ribosomal leaky scanning that initiates translation from internal in-frame AUG codons corresponding to methionines M20, M53, M69 and M83 of P1 [13]). P1 alone contains residues 1–19 that are required for association with the viral L-protein so that P1 can act as the polymerase cofactor [18]. All P-protein isoforms contain the CTD which incorporates the binding sites for viral genome–associated N-protein (N-RNA), [17,23–25] and STAT1 (black boxes) [11,19,26]. P-protein interaction with EXPs and IMPs is mediated through two CRM-1-binding NESs (yellow boxes) [27,28] and two IMPα/β-binding NLSs (blue boxes, described elsewhere [9,27] and in this study), located in both the NTR and CTD. A third potential NES has been suggested to exist within NTR residues 53–174 [9]. The NTR-localized N-NLS is activated in P3 due to truncation of residues 1–52, which also truncates/deactivates the N-NES [9,27]. The P-CTD localized C-NLS, which includes key residues K214/R260 (blue boxes) of a positively charged patch on the surface of the CTD, is characterized in this study; the positive patch has also been implicated in binding to N-RNA [19,24,29–31], suggesting that the N-RNA-binding site overlaps with the C-NLS [24,25,27,28]. The nucleocytoplasmic (Nuc/Cyt) localisation of each isoform is indicated (cytoplasmic (C), nuclear (N), diffuse (N/C)).
Fig 2.
Mutation of K214/R260 affects nuclear localization of P3 through a mechanism involving active nuclear export and impaired nuclear import.
(A) COS-7 cells transfected to express the indicated proteins were treated with or without LMB (2.8 ng/ml, 3 h) prior to analysis of living cells by CLSM. (B) Images such as those shown in A were analyzed to determine the ratio of nuclear to cytoplasmic fluorescence (Fn/c) corrected for background fluorescence for individual cells as previously described [9,28]; the histogram shows the mean Fn/c ± S.E.M., calculated for n ≥ 30 cells for each transfection/condition. Data are representative of three separate assays. Statistical analysis used Students t-test (**, p ≤ 0.01; ****, p < 0.0001).
Fig 3.
Mutation of K214/R260 prevents P1 nuclear accumulation.
(A) COS-7 cells transfected to express the indicated proteins were treated without or with LMB (2.8 ng/ml, 3h) before imaging of living cells by CLSM and (B) determination of Fn/c as described in the legend to Fig 2 (mean ± S.E.M., n ≥ 50 cells). Data are representative of three separate assays; statistical analysis used Student’s t-test: **** p < 0.0001)
Fig 4.
P-CTD mediates nuclear import of GFP that is inhibited by mutation of K214/R260.
(A) Purified recombinant GFP-T-ag NLS, GFP-P-CTD, and GFP-P-CTD-K214/R260-A protein was added to mechanically perforated HTC cells together with exogenous cytosol (rabbit reticulocyte lysate) and an ATP-regeneration solution to reconstitute nuclear import, and TR70 to monitor nuclear envelope integrity [39,40]. Nuclear localization of the GFP-fused proteins was then monitored over 30 min by CLSM (representative images at 2 and 30 min are shown). (B) Images such as those shown in A were analyzed to determine the Fn/c as described in the legend to Fig 2 (mean ± S.E.M., n ≥ 3 nuclei at each timepoint). Data are from a single assay representative of two separate assays. Curve fitting used GraphPad Prism 6 (one-phase association).
Fig 5.
P-CTD mediates nuclear import of GFP dependent on IMPα2 and IMPβ1.
Nuclear import of purified GFP-P-CTD and GFP-T-ag NLS was analyzed as described in the legend to Fig 4 except that exogenous cytosol was pre-incubated (15 min) with the indicated antibodies. (A) CLSM images following 30 min incubation of GFP-P-CTD and GFP-T-ag NLS in the presence of the indicated antibody are shown. (B) Images such as those in A were analyzed to determine the Fn/c as described in the legend to Fig 2 (mean ± S.E.M., n ≥ 3 nuclei for each time point; data are from a single assay typical of three separate assays). Curve fitting used GraphPad Prism 6 (one-phase association).
Fig 6.
P-CTD interacts with IMPα2, IMPβ1 and IMPα2/β1 dependent upon K214/R260.
(A) 2 μM GFP-fused wt P-CTD, P-CTD-K214/R260-A, TRF1 or T-ag NLS was incubated with 10 μM GST-fused mouse ΔIBB-IMPα2, IMPβ1 or IMPα2/β1 before native gel electrophoresis and fluoroimaging as previously described [9,39,42]. Binding to IMPs is indicated by altered mobility of the GFP-fused proteins compared to that of no IMP controls (different complexes are indicated by arrows). Data is from a single assay representative of two independent assays. (B, C) 30 nM His6-tagged GFP-P-CTD wt or GFP-P-CTD-K214/R260-A was incubated with increasing concentrations of (B) biotinylated-IMPα2/β1 or (C) biotinylated-IMPβ1 prior to AlphaScreen analysis. Assays were performed in triplicate; graphs show mean AlphaScreen counts ± S.E.M. (n = 3) from a single typical experiment, representative of four independent assays.
Fig 7.
IMPα2 co-precipitates with RABV P-proteins from mammalian cells, dependent on K214/R260.
HEK293T cells expressing the indicated GFP-fused proteins were subjected to immunoprecipitation using GFP-Trap before analysis of immunoprecipitate (IP) and whole cell lysate (WCL) by immunoblotting (IB) for IMPα2 and GFP. Data is from a single assay, representative of three independent experiments.
Fig 8.
Model of rabies P protein isoform nuclear trafficking.
(A) P1 and P2 localize to the cytoplasm due to the activity of the strong N-NES together with the C-NES (yellow rectangles and circles, respectively) which counters nuclear import mediated by the C-NLS alone (blue circle) [27,28]. (B) P3 accumulates within the nucleus via import driven by the N-NLS [9] (blue rectangle) and C-NLS. (C) Nucleocytoplasmic localization of P4-5 is diffuse, due trafficking driven by the C-NLS and C-NES only. Transport is indicated by arrows, with thicker arrows indicating the predominant direction.