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First published online 10 June 2003
doi: 10.1242/jcs.00528

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The role of IFN nuclear localization sequence in intracellular function

Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL, 32611-0700, USA

View larger version (20K): [in a new window]   Fig. 1. Synthesis and intracellular retention of IFN. (A) WISH cells, untreated (lanes 1 and 2) or those transduced for two days with an empty vector control (lanes 3 and 4) or a vector expressing non-secreted IFN (lanes 5 and 6) or a vector expressing non-secreted IFN mutated in the NLS (lanes 7 and 8) were used. Proteins from cell extracts (odd-numbered lanes) or supernatants (even-numbered lanes) were separated by SDS-PAGE and probed with an antibody to IFN. Detection was carried out by using chemiluminescence. (B) Quantitation of IFN produced in L929 cells by ELISA. Cell extracts (odd numbers) and supernatants (even numbers) from L929 cells, transduced for two days with the empty adenoviral vector (column 1 and 2) or vector expressing non-secreted IFN (column 3 and 4) or vector expressing non-secreted IFN mutated in the NLS (column 5 and 6) were assayed for IFN by ELISA.

View larger version (11K): [in a new window]   Fig. 2. Resistance to viral infection by intracellular human IFN is dependent on the presence of the NLS. Mouse L929 cells, untreated (column 1) or those transduced for 1 hour with an empty vector control (column 2), a vector expressing non-secreted human IFN (column 3) or a vector expressing non-secreted IFN with a mutation in the NLS (column 4) were allowed to grow for 24 hours, followed by infection with VSV for 24 hours. Cells were then stained with crystal violet, extracted with methylcellusolve, and the absorbance was measured, as described in Materials and Methods. Absorbance in cells that were not exposed to any virus was taken as 100%, and the percentage of cells surviving other treatments is presented. Results represent the mean of three independent determinations.

View larger version (17K): [in a new window]   Fig. 3. Induction of MHC class I by intracellular expression of IFN is abolished by removal of the NLS. WISH cells were transduced for 1 hour with an empty vector control (dotted line), a vector expressing non-secreted IFN (dark grey) or a vector expressing non-secreted IFN mutated in NLS (light grey). Cells were then allowed to grow for 48 hours in regular medium followed by staining with R-PE-conjugated monoclonal antibody to human MHC class I and analysis by flow cytometry. R-PE-conjugated murine IgG2a was used as a control. A similar profile was noted in three independent experiments.

View larger version (28K): [in a new window]   Fig. 4. Phosphorylation of STAT1 by intracellular IFN is independent of the IFN C-terminal NLS. WISH cells, untreated (lane 1) or treated with an empty vector control (lanes 2), a vector expressing non-secreted IFN (lanes 3) or a vector expressing non-secreted IFN with a mutated NLS (lanes 4) were allowed to grow for 8 hours. Proteins from whole cell extracts were electrophoresed and probed with an antibody to phospho-STAT1 (upper panel). Filter was stripped and re-probed with an antibody to STAT1 (lower panel). Detection was by chemiluminescence.

View larger version (39K): [in a new window]   Fig. 5. Association of activated STAT1, IFN and IFNGR1 with nuclear importer, NPI-1. Cell extracts from WISH cells transduced for 8 hours with an empty vector (lane 1), NLS-mutated IFN expression vector (lane 2) or non-secreted IFN expression vector (lane 3) were used for immunoprecipitation with an antibody to NPI-1. Equal amounts of immunoprecipitates were electrophoresed and probed individually with antibodies to IFNGR1 (first row), IFN (second row), phospho-STAT1 (third row) or NPI-1 (fourth row).

View larger version (97K): [in a new window]   Fig. 6. Nuclear translocation of STAT1 and IFNGR1 by intracellular IFN requires the NLS of IFN. (A) WISH cells were transfected for 8 hours with an empty vector (column 1), a vector expressing non-secreted IFN (column 2) or a vector expressing NLS-modified IFN (column 3) and stained simultaneously with antibodies to STAT1 and IFNGR1. Secondary antibodies to STAT1 conjugated to Alexa 594 (top row) or to IFNGR1 conjugated to Cy-2 (bottom row) were used and analyzed by fluorescence microscopy. (B) Quantitation of images. Images of cells transduced with an empty vector control (left lanes), a vector expressing non-secreted IFN (middle lanes) or a vector expressing NLS-mutated IFN (right lanes) were viewed in seven different fields to a obtain mean ratio of nuclear pixel intensity (Fn) to cytoplasmic pixel intensity (Fc). STAT1 and IFNGR1 Fn/Fc for nsIFN versus mutant were both significant at P<0.002 by t-test. Calculations for fluorescence in the nucleus (N) versus cytoplasm (C) were also done by using N/N+C. Four independent measurements showed a P<0.03 by t-test for the nuclear translocation of STAT1 and IFNGR1 for the wild-type IFN versus the NLS-mutated IFN.

View larger version (32K): [in a new window]   Fig. 7. Nuclear translocation of IFN requires the presence of the C-terminus NLS. (A) WISH cells, transduced for 8 hours with an empty vector control (top) or a vector expressing non-secreted IFN (middle left) or NLS-modified IFN (middle right) were probed with a monoclonal antibody to IFN and stained with a Alexa-Fluor-488-conjugated secondary antibody and analyzed by confocal microscopy. The lower graphs show mean fluorescence intensity comparisons for non-secreted IFN and NLS-mutated IFN. Measurements of fluorescence intensities were done with IP Lab (Scanalytics) using the MP-line measure tool. Mean pixel intensity was measured across a line drawn through the cells in the plane of the images shown. A representative measurement line is shown in white. The resultant graphs were generated by the software averaging the intensities of a total of 100 pixels on either side of the line at each point along the line. Triplicate determinations showed a significance of P<0.25 (t-test) for nuclear presence of IFN versus NLS mutant. (B) Quantitation of images. Images of cells transduced with non-secreted IFN or NLS-mutated nonsecreted IFN were used to determine Fn/Fc values, which are shown in columns 1 and 2, respectively. Seven fields were examined and the results were averaged±s.d. The significance was P<0.02 by the t-test. Calculations for fluorescence in the nucleus (N) versus cytoplasm (C) were also done by using N/N+C. Four independent measurements showed a P<0.025 for the nuclear translocation of IFN for the wild-type IFN versus the NLS-mutated IFN.

View larger version (23K): [in a new window]   Fig. 8. Intracellular presence of peptide IFNGR-1(253-287) inhibits binding to IFNGR of extracellular IFN and subsequent activation of STAT1. (A) Presence of extracellular peptide IFNGR-1(253-287) did not inhibit binding of 125I-IFN to P388D1 cells at the concentrations to be used in subsequent experiments. Unlabeled murine IFN or peptide IFNGR-1(253-287), as indicated, was added at a final concentration of 1 µM to P388D1 cells at 4°C along with 10 nM of 125I-IFN, and cells were incubated at 4°C for 30 minutes. Control cells were incubated with 125I-IFN in the absence of any competitor. Cells were then washed and bound IFN determined. Samples were run in triplicate and values plotted as mean±s.d. (B) Intracellular accumulation of peptide IFNGR-1(253-287) in P388D1 cells by pinocytosis was accomplished by incubating cells with either 25 µM (lane 2) or 50 µM (lane 3) of peptide at 37°C for 1 hour. Cells used in lanes 1 and 4 did not receive any peptide. Cells were then washed at room temperature to remove extracellular peptide and then incubated with 125I-IFN (10 nM) along with 1 µM of IFNGR-1 peptide for 5 minutes at 37°C. Control cells (lane 1) were washed in ice-cold medium and then incubated with 125I-IFN at 4°C without peptide. After 125I-IFN incubation, all cells were washed at 4°C and then acid-washed at 4°C to remove surface-bound 125I-IFN. Cells were then lysed and immunoprecipitated with antibodies to IFNGR-1. After western transfer of immunoprecipitates to nitrocellulose membranes, 125I-IFN associated with IFNGR-1 was detected by autoradiography. Total IFNGR-1 immunoprecipitated was followed by immunodetection with IFNGR-1 antibodies (lower panel). (C) Conditions are the same as in (B), except that lysates were immunoprecipitated with STAT1 antibodies and tyrosine phosphorylation of immunoprecipitated STAT1 was followed by immunodetection with antibodies specific for Tyr701-phosphorylated STAT1. Total immunoprecipitated STAT1 was followed by reprobing blots with antibodies to STAT1 (lower panel).

View larger version (16K): [in a new window]   Fig. 9. The deletion mutant IFN(1-123) is not internalized compared to wild-type IFN. Internalization assays were run as described previously (Farrar et al., 1991). Briefly, 125I-labeled wild-type and mutant IFN (80-85 µCi/µg) were added to WISH cells at 5 nM. Cells were incubated at 4°C for 90 minutes, washed at 4°C and then incubated at 37°C for the indicated times. At the indicated times internalized cpm was counted from acid-washed cells lysed in 1% SDS. Data are plotted as a percentage of the total ligand bound that was internalized. Samples were run in triplicate and data are plotted as mean±s.d.