Figure 1.
ERK activation is sustained in de novo RRV infection.
(A) Quantitative immunoblots of uninfected (UI; left panels), UV irradiated-RRV infected (UV-RRV; MOI of 10 pre UV treatment; middle panels), or RRV-infected RhF (MOI of 10; right panels). 10 ug of cell lysate (10–15% of total lysate) was loaded per lane. Blots were probed with the following antibodies: anti-phosphorylated ERK (pERK), anti-Ran (loading control for cellular extracts), and anti-ERK1/2. (B) Graphical representation of the fold change in pERK2 over time relative to time zero. pERK2 values were normalized to Ran to correct for loading differences. Normalized pERK2 values were compared to time zero for each of the three conditions to determine the fold change. Two representative experiments are plotted; solid and dashed lines symbolize two separate experiments. Green, blue, and red lines represent UI, UV-RRV, and RRV respectively.
Figure 2.
Nuclear localization of pERK with persistent phosphorylation of nuclear and cytoplasmic ERK targets.
(A) Immunofluorescence images of UI (i) and infected (MOI of 5) (ii, iii) RhF. Cells were fixed, permeabilized, and co-stained with DAPI (blue) and pERK (red) and the two images were merged. Enlarged images from ii and iii are shown to the right of these images. (B) Immunoblot analysis of UI (lane 1) or infected (lane 2, MOI of 10) RhF collected 48 h p.i. Blots were probed with both phospho-specific and total antibodies for: RSK1/2 (top panels), MNK-1 (middle panels), Elk-1 (3rd panels), and Ran to normalize for loading differences.
Figure 3.
RRV ORF45 (R45) expression leads to the phosphorylation of ERK.
(A) Immunoblots of 293 cells transfected with empty vector (lane 1), HA-RSK2 (lane 2), FLAG-R45 (lane 3), or co-transfected with FLAG-R45 and HA–RSK2 (lane 4) expression vectors. Cells were collected 48 h post-transfection and proteins from whole cells lysates were analyzed by quantitative immunoblotting (10–15% of total lysate was loaded per lane). Blots were probed with antibodies to detect the indicated proteins. Asterisk (*) above ERK2 represents pERK2. (The original blots from which this figure was derived is shown in Figure S8.) (B) Graphical representation of the fold change in pERK levels in 293 cells transfected with either vector alone (lane 1), FLAG-R45 (lane 2), or FLAG-R45/HA-RSK2 (lane 3). Data are representative of 3 independent experiments for pERK1 and 6 independent experiments for pERK2. P values were determined using Student's t test comparing the experimental condition to the control values. Asterisks above the bars denote the level of statistical significance. In comparing pERK1 levels from vector to R45 alone or to R45/RSK2, p values were 0.2005 and 0.0059 respectively; in comparing pERK1 levels from R45 to R45/RSK2, p value was 0.2264. In comparing pERK2 levels from vector to R45 alone or to R45/RSK2, p values were 0.0059 and 0.0006 respectively; in comparing pERK2 levels from R45 to R45/RSK2, p value was 0.0481.
Figure 4.
R45 forms a complex with ERK and RSK.
(A) Immunoblots of RhF uninfected (lane 1) or infected with RRV (lane 2; MOI of 10) for 48 h. Blots were probed with the indicated antibodies. Whole cells lysates (10–15% of total lysate was loaded per lane) from lane 2 were immunoprecipitated with either rabbit IgG as a negative control (lane 3) or anti-R45 (lane 4). The immunoprecipitates were blotted with the antibodies indicated. (B) Immunoblots of 293 cells transfected with empty vector alone (lane 1) or co-transfected with FLAG-R45 and HA-RSK1 (lane 2) or HA-RSK2 (lane 3) and harvested 48 h after transfection. Cell lysates were immunoprecipitated with anti-FLAG affinity gel and eluted with 3× FLAG peptide (10–15% of total lysate was loaded per lane). The eluates were then sequentially immunoprecipitated with anti-HA affinity gel. Whole cell lysates and immunoprecipitates were analyzed by immunoblotting and probed with the antibodies indicated to the right of the blots. Starred bands (*) in lanes 2 and 3 represent pERK2.
Figure 5.
R45 interacts with pERK and RSK2 during de novo RRV infection.
(A) Images of 293 cells transfected with FLAG-R45 (top row), HA-RSK2 alone (2nd row), or FLAG-R45 and HA-RSK2 (3rd row). As controls, cells were transfected with either an empty FLAG vector (pCMV-Tag3a; 4th row) or empty HA vector (pKH3; bottom row). The images in the 4th column represent a merge of columns 2 and 3. (B) RhF infected with RRV (MOI of 5) for 48 h were fixed, permeabilized, and stained with the indicated antibodies, R45, pERK, and RSK2. The inset in each single stained image, denoted by a white box, was merged (columns 2–4 only) to produce the overlay (right-most panel). An enlarged image (4×) of the merged boxed area is shown below. Original magnification of 63×.
Figure 6.
The R45-containing complex predominantly protects pERK2 over pERK1.
(A) Quantitative immunoblots of RRV-infected RhF (48 h; MOI of 2.5) treated with DMSO (left) or the MEK inhibitor, U0126 (50 uM, [right]). 10–15% of total lysate was loaded per lane. Blots were probed with antibodies to phosphorylated ERK (pERK) and GAPDH. Times indicated above blots represents hours after U0126 treatment. (B) Graphical representation of decay of GAPDH normalized pERK1 and pERK2 levels following the addition of DMSO or U0126. Data are the mean of three independent experiments with error bars reflecting the SEM. R2 values indicate the coefficient of determination for each 1st order exponential decay curve. (Of note, the lower MOI of 2.5 helped minimize the degree of lysis between p.i. hours 48 to 58).
Figure 7.
FRET analysis of RRV infected RhF reveals R45 in close proximity to both pERK2 and pRSK2 within the nuclei, but not the cytoplasm.
(A) 1st column: Representative uncorrected (without background subtraction) FRET images of RRV-infected RhF stained with (top) anti-pERK (AF 488, donor) and anti-R45 (AF 555, acceptor) or (bottom) anti-R45 (AF488, donor) and anti-RSK2 (AF 555, acceptor). 2nd column: software-generated regions of interest/ROIs (from 1st column) used for precise FRET (PFRET) analysis (see below). 3rd column: Merged confocal images from the same fields as in first two columns. 4th column: Enlarged images of the cytoplasm (4.4× magnification) from boxed region in 3rd column. Arrows indicate examples of nuclei where a FRET signal is present and proteins co-localize by standard confocal microscopy. (B) Acceptor photobleaching (AP) FRET from RRV infected cells (MOI of 5). The acceptor was bleached for each region of interest (ROI; see also (A) above, column 2) within the nuclei (blue diamonds) and cytoplasm (red diamonds) to determine the efficiency of energy transfer. Efficiency of energy transfer was calculated for each point using the following equation: E% = (Donor post bleach - Donor prebleach)/Donor post bleach. E% less than 5% was discounted as background.
Figure 8.
RSK is not required for ERK activation.
Immunoblots of RhF transfected with non-targeting siRNA (siCNL) or sRSK1+siRSK2, as indicated. (A) 24 h post-transfection cells were infected with RRV (MOI of 5) and then harvested at 48 h p.i. Cell lysates were probed with the indicated antibodies (10–15% of total lysate was loaded per lane). The quantitative immunoblot shown is representative of three separate experiments. (Of note, the original blot from which these two lanes were derived is shown in Figure S9.) Graphical representation of the expression of (B) pRSK (pRSK2, T573 antibody); compared to siCNL, pRSK levels in the siRSK condition were statistically different; p = 0.0005 or (C) pERK isoforms treated with siRSK1 plus siRSK2 or siRNA control; compared to siCNL, pERK1 and pERK2 levels in the siRSK condition were statistically different (p = 0.0024 and 0.0014. respectively). (D) Quantitative immunoblot of whole cell lysates from a separate siRSK experiment was probed with antibodies to two structural viral proteins, MCP (top panel) and R45 (middle panel) and normalized to GAPDH (bottom panel) to correct for loading (10–15% of total lysate was loaded per lane). (E) Graphical representation of (D); compared to siCNL, R45, but not MCP, levels in the siRSK condition were statistically different (p = 0.0042 and 0.38, respectively). (F) Viral titers in the supernatants from cells in (A); compared to siCNL, titers from supernatants in the siRSK condition were not statistically different (p = 0.50). For (A) through (F), data represent the mean +/− SEM of 3 separate experiments. P values were determined using Student's t test comparing the experimental and control conditions. Columns lacking asterisks were statistically indistinguishable from corresponding siCNL values.