Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3

This information is current as Mingzhu Zhu, Ting Fang, Shun Li, Kun Meng and Deyin of September 27, 2021. Guo J Immunol published online 20 May 2015 http://www.jimmunol.org/content/early/2015/05/20/jimmun ol.1500232 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 20, 2015, doi:10.4049/jimmunol.1500232 The Journal of Immunology

Bipartite Nuclear Localization Signal Controls Nuclear Import and DNA-Binding Activity of IFN Regulatory Factor 3

Mingzhu Zhu, Ting Fang, Shun Li, Kun Meng, and Deyin Guo

Accurate cellular localization plays a crucial role in the effective function of most signaling , and nuclear trafficking is central to the function of transcription factors. IFN regulatory factor (IRF)3 is a master responsible for the induction of type I IFN, which plays a crucial role in host antiviral innate immune responses. However, the mechanisms for control and regulation of IRF3 nuclear import largely remain to be elucidated. In our study, we identified a bipartite nuclear localization signal (NLS) in IRF3, with two interdependent basic clusters separated by a 7-aa linker. Our study further demon- strated that the bipartite NLS of IRF3 is also critical for IRF3 DNA-binding activity, indicating that the two functions of this region are integrated, which is in contrast to other IRFs. Furthermore, the IFN bioassay and infection studies suggest that IRF3 NLS is

essential to the IRF3-mediated IFN responses and antiviral immunity. Overall, our results reveal a previously unrecognized bi- Downloaded from partite NLS for IRF3 that contains both DNA-binding activity and nuclear import function, and they shed light on the regulatory mechanisms of IRF3 activation and IRF3-mediated antiviral responses. The Journal of Immunology, 2015, 195: 000–000.

nnate immunity contributes to the first line of host defense inflammation, and apoptosis (15). All of these members are most against microbial infection. Host cells have evolved a variety conserved in their N-terminal DNA-binding domain (DBD), which I of mechanisms to recognize and eliminate invading patho- recognizes the IFN-stimulated response element (ISRE) (consensus http://www.jimmunol.org/ gens. Recognition of pathogen is primarily mediated by pattern sequence: 59-GAAANNGAAAG/CT/C-39), as found in the PRD and recognition receptors, including TLRs, RIG-I–like receptors, and PRD-LEs of the IFN-a/b promoters (16, 17). Among IRFs, at least NOD-like receptors, which invoke powerful cellular signaling four members, IRF1, IRF3, IRF5, and IRF7, have been implicated cascades to stimulate the production of various (1). Upon as positive regulators of IFN-a/b transcription (18). IRF1 is virus infection, cytoplasmic RIG-I–like receptors and endosomal the first identified IRF member and activates the type I IFN gene TLRs sense viral RNAs or DNAs and trigger rapid production of type promoter (19). However, the induction of IFN-a/b mRNAs was I IFN (IFN-a/b), which confer resistance to virus infection through normal in virus-induced Irf12/2 or Irf52/2 mouse embryonic fibro- the induction of hundreds of IFN-stimulated (2–4). Thus, type blasts (MEFs) (20, 21), which indicates that neither IRF1 nor IRF5 I IFN plays an essential role in the host innate antiviral response. is essential for the induction of IFN-a/b gene. IRF3 and IRF7, by guest on September 27, 2021 The transcription activation of type I IFN has been studied which are highly homologous, were shown to play an essential role extensively. The IFN-b gene promoter region contains at least four in the virus-induced expression of the type I IFN gene (22). regulatory cis-elements: the positive regulatory domains (PRDs) I, IRF3 is expressed constitutively in all cell types and resides in II, III, and IV (5–8). The promoter region of IFN-a4 gene contains the cytoplasm prior to virus infection. Upon virus infection, it PRD I and PRD III–like elements (PRD-LEs), which bind IFN undergoes C-terminal phosphorylation and dimerization and then regulatory factor (IRF) family members (9–11). The PRD I and translocates into the nucleus, subsequently forming a complex with PRD III elements are activated by members of the IRF family, the coactivators CBP/p300 and binding to the PRD and PRD-LEs of whereas the PRD II and PRD IV elements are recognized by NF- the IFN-a/b promoters and ISRE sequences of targeted genes, kB and ATF-2/c-Jun, respectively (12–14). including the cytokines RANTES and IP-10 (23–25). The bio- The IRF family contains nine mammalian members that play logical effect of IRF3 transcriptional activity requires its ability to a critical role in diverse biological processes, including immunity, translocate into the nucleus. For this reason, the nuclear–cyto- plasmic shuttling is a critical aspect of its regulation. Eukaryotic cells have evolved with the genome restricted to the State Key Laboratory of Virology and Modern Virology Research Center, College of membrane-bound nucleus, which is separated from the cytoplas- Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China mic compartment. This separation coevolved with the regulatory Received for publication February 5, 2015. Accepted for publication May 3, 2015. mechanisms that control the nuclear–cytoplasmic trafficking of This work was supported by the National Basic Research Program of China proteins and RNAs. Small molecules can diffuse through the nu- (2010CB911800) and the National Natural Science Foundation of China (81130083 and 31221061). D.G. receives support from Hubei Province’s Outstanding Medical clear pore complexes, but the passage of proteins . 40 kDa relies Academic Leader Program. on soluble receptors and carrier molecules that mediate move- Address correspondence and reprint requests to Dr. Deyin Guo, College of Life Sci- ment across nuclear pore complexes (26). The passport-recognition ences, Wuhan University, Wuhan 430072, People’s Republic of China. E-mail address: signals on cargo are amino acid sequences corresponding [email protected] to either nuclear localization signals (NLSs) or nuclear export The online version of this article contains supplemental material. signals (NESs) (27). Typical NLSs contain one (monopartite) or Abbreviations used in this article: DBD, DNA-binding domain; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; MEF, mouse embryonic fibroblast; two (bipartite) clusters of basic residues. Monopartite NLSs, such NES, nuclear export signal; NLS, nuclear localization signal; PRD, positive regula- as that of SV40 large-T Ag, have a single cluster of four or five tory domain; PRD-LE, PRD I and PRD III–like element; SeV, Sendai virus; VSV, basic residues (28), whereas bipartite NLSs, exemplified by nucle- vesicular stomatitis virus; VSV-GFP, GFP-containing VSV; WT, wild-type. oplasmin, have a second basic cluster located ∼10–12 residues Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 downstream of the first cluster (29). The subcellular distribution

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500232 2 CHARACTERIZATION OF IRF3 BIPARTITE NLS of IRF family members has been investigated, and the key amino erase activities in the total cell lysates were measured with a dual-specific acids responsible for their nuclear import have been determined luciferase reporter assay system (Promega). Firefly luciferase activities for most IRFs. However, the nature and function of the NLS of were normalized on the basis of Renilla luciferase activities. The relative luciferase activity of the IFN-b promoter was a ratio of the readout value IRF3 have not been adequately characterized, and little is known of firefly/Renilla luciferase activity for each sample. All reporter assays about that of IRF7. were repeated at least three times. Data shown are mean 6 SD from one In IRF3, a leucine-rich sequence (ILDELLGNMVL) was iden- representative experiment. tified to function as an NES spanning aa 139–149 (25). A previous Subcellular fractionation study defined the general localization of the IRF3 NLS domain but lacked a detailed characterization of it (30). In our study, we Nuclear and cytoplasmic extracts were prepared using the Nuclear-Cytosol Extraction Kit (Applygen Technologies), according to the manufacturer’s demonstrated that IRF3 contains a bipartite NLS that controls both instructions. DNA-binding activity and nuclear import. First, analysis of GFP- IRF3 fusion proteins shows that IRF3 contains the necessary Protein expression and purification sequences within or flanking the DBD (aa 64–130) to specify its IRF3 1–115 or IRF3 1–115, coupled with mutation KR77/78NG, R81L, or nuclear import. Further mutagenesis determined the basic amino RK86/87LQ coding sequences, was inserted in frame to the GST coding acids responsible for IRF3 nuclear import. Our studies showed sequence in the bacterial expression vector pGEX-6P-1. Escherichia coli BL21 (DE3) cells (500 ml at OD600 nm = 1.2) harboring the recombinant that the bipartite NLS of IRF3 is characterized by the basic amino b 77 78 86 87 expression vectors were incubated with 0.4 mM isopropyl- -D-thio- acid clusters KR and RK , which may occupy the minor galactopyranoside at 16˚C for 24 h. Cells were harvested by centrifugation binding site and major binding site of importin-a, respectively. and suspended in 5 ml lysis buffer containing 50 mM Tris-HCl (pH 7.6),

Mutation of 77KR78 partially abrogates the nuclear import ability, 1 mM EDTA, 2 mM DTT, 0.1 mM PMSF, 0.02% Nonidet P-40, 2 mM Downloaded from m whereas mutation of 86RK87 totally disrupts the nuclear import abil- leupeptin, 10 mM MgCl2,150mMNaCl,0.4mg/mllysozyme,and40 g/ml DNase I. Cells were lysed for 60 min. A total of 5 ml bacterial supernatant ity. Immunofluorescence imaging assays reinforced our results. Our was mixed with 200 ml 50% (v/v) glutathione-agarose beads and incubated study further demonstrated that the bipartite NLS of IRF3 is also for 60 min at 4˚C with gentle rotation. The agarose beads were washed critical for IRF3 DNA-binding activity, indicating that the two four times with 5 ml ice-cold PBS. The recombinant proteins were cleaved functions of this region are integrated. Overall, our results revealed and eluted through incubation with 100 ml 50 mM Tris (pH 8), 2 mM DTT, and GST-tagged human rhinovirus (HRV 3C) protease (50 U) at 4˚C for 5 h. a previously unrecognized bipartite NLS for IRF3 that contains both http://www.jimmunol.org/ Supernatants containing the recombinant proteins were collected by cen- DNA-binding activity and nuclear import ability, which is unique trifugation. The purity and quantity of the recombinant proteins were ex- among the IRF family proteins. amined by SDS-PAGE, followed by Coomassie blue staining. EMSAs Materials and Methods Cell culture and reagents For the IRF3:DNA complex, binding reactions were assembled at 21˚C in a total volume of 5 ml in buffer 10 mM HEPES (pH 8), 500 mM NH4OAc, 2/2 2/2 Irf3 Irf7 MEFs were provided by Dr. Ping-hui Feng (University of 100 mM NaCl, 5 mM MgCl2, 5% glycerol, and 1 mM DTT. Binding Southern California, Los Angeles, CA). Cells were grown in DMEM reactions were loaded onto an actively running 7% polyacrylamide gel in supplemented with 10% FCS. Sendai virus (SeV) and GFP-containing ve- 0.53 TBE (45 mM Tris, 45 mM borate, 1 mM EDTA [pH 8.3]) that had sicular stomatitis virus (VSV) (VSV-GFP) were supplied by Dr. Hong-Bing been pre-electrophoresed for 30 min at 4˚C. Electrophoresis continued for by guest on September 27, 2021 Shu (Wuhan University). Mouse monoclonal anti-Flag, anti-HA (Sigma), an additional 60 min at 4˚C after which the gel was stained with 0.53 TBE rabbit polyclonal anti-IRF3, anti-LMNB1, anti-GAPDH, and anti-tubulin containing 0.5 mg/ml ethidium bromide. Samples were resolved on a 7% (Proteintech) were purchased from the indicated manufacturers. native gel composed of 49:1 acrylamide:bis-acrylamide in a 0.53 TBE- based buffer. Constructs Probe sequences used were as follows: IFNB1 sense: 59-TAAATGA- CATAGGAAAACTGAAAGGGAGAAGTGAAAGTGGGAAATTCCTCT- b The IFN- promoter luciferase reporter plasmid was provided by Dr. Hong- GAATAG-39;antisense:59-ACTATTCAGAGGAATTTCCCACTTTCACT- Bing Shu. Mammalian expression plasmids for GFP-tagged IRF3 and its TCTCCCTTTCAGTTTTCCTATGTCATTT-39. truncated mutants were constructed by standard molecular biology tech- The DNA probe was mutated to make the IRF3-binding sites deficient, niques. and the mutated probe sequences used were as follows: IFNB1 mutated 9 Site-directed mutagenesis technique sense: 5 -TAAATGACATAGGCCCCCTCCCCGGCCACCGTCCCCGT- GGGAAATTCCTCTGAATAG-39; antisense: 59-CTATTCAGAGGAATT- The site-specific mutants of IRF3 were constructed using the site-directed TCCCACGGGGACGGTGGCCGGGGAGGGGGCCTATGTCATTTA-39 mutagenesis kit (Vazyme). The primers for the mutations are as follows: RK68/70SQ Fwd: 59-GCATATGTTCCCGGGAGCGATCAGCCAGACC- Immunofluorescent labeling and confocal microscopy 9 9 TG-3 ; RK68/70SQ Rev: 5 -GATCGCTCCCGGGAACATATGCACCAG- The cells were fixed with 4% paraformaldehyde in PBS for 10 min and then TGGCC-39; KR77/78NG Fwd: 59-CCTGCCAACCTGGAACGGGAATT- permeabilized with 0.2% Triton X-100 in PBS for 15 min. After blocking TCCGC-39; KR77/78NG Rev: 59-CGTTCCAGGTTGGCAGGTCTGGCT- cells with 1% BSA in PBS for 30 min, the coverslips were incubated with TATC-39;R81LFwd:59-CTGGAAGAGGAATTTCCTCTCTGCCCTCA- the indicated primary Abs and FITC-goat anti-rabbit IgG or TRITC-goat AC-39; R81L Rev: 59-AGGAAATTCCTCTTCCAGGTTGGCAGGTC-39; 9 9 anti-mouse IgG sequentially, and observed with a Leica confocal micro- RK86/87LQ Fwd: 5 -CTGCCCTCAACCTCCAAGAAGGGTTGCG-3 ; scope using a 3100 oil-immersion objective. RK86/87LQ Rev: 59-GGAGGTTGAGGGCAGAGCGGAAATTC-39;R91L Fwd: 59-CAAAGAAGGGTTGCTTTTAGCAGAGG-39;R91LRev:59-AG- Detection of production CAACCCTTCTTTGCGGTTGAGGGC-39; RK96/98SQ Fwd: 59-GTTTAG- CAGAGGACCTGAGCCAGGACCCTCACG-39; RK96/98SQ Rev: 59-GG- The concentrations of IFN-b in culture supernatants were measured with CTCAGGTCCTCTGCTAAACGCAACCCTTC-39; K105Q Fwd: 59-CAA- a mouse IFN-b ELISA kit (BioLegend), according to the manufacturer’s AGAAGGGTTGCTTTTAGCAGAGG-39; and K105Q Rev: 59-AGCAAC- instructions. CCTTCTTTGCGGTTGAGGGC-39. VSV plaque assay Transfection and reporter gene assays Irf32/2 Irf72/2 MEFs (2 3 105) were transfected with the indicated MEFs were seeded on 24-well plates (1 3 105 cells/well) and transfected plasmids for 24 h prior to VSV infection. At 1 h postinfection, cells were the following day with 50 ng IFN-b firefly luciferase reporter plasmid washed with PBS three times, and fresh medium was added. The super- together with a total of 0.8 mg expression plasmid or empty control natants were harvested at 24 h, diluted 1:106–1:107, and used to infect plasmid via standard calcium phosphate precipitation or Lipofectamine confluent BHK21 cells plated on 24-well plates. At 1 h postinfection, the 2000 (Invitrogen). To normalize for transfection efficiency, we added 20 ng supernatants were removed, and medium containing 0.75% agar was pRL-TK Renilla luciferase reporter plasmid to each transfection. Lucif- overlaid. At 2–3 d postinfection, cells were fixed with 4% formaldehyde The Journal of Immunology 3 for 20 min and stained with 0.2% crystal violet. Plaques were counted, shown in Fig. 1B, in uninfected cells, IRF3 localized exclusively averaged, and multiplied by the dilution factor to determine viral titers to the cytoplasm; SeV infection resulted in translocation of IRF3 (PFU/ml). to the nucleus within 12 h. These results are consistent with Native-PAGE previous observations (31). IRF3 contains both NLS and NES, and the predominant cyto- Native-PAGE was performed using an 8% acrylamide gel without SDS. The gel was prerun with 25 mM Tris-HCl (pH 8.4) and 192 mM glycine, with or plasmic localization of IRF3 before virus infection implies that the without 0.5% deoxycholate, in a cathode and anode chamber, respectively, NES is constitutively active, and nuclear export is more dominant for 30 min at 40 mA on ice. Samples in the native sample buffer (50 mM than nuclear import. To define the NLS domain of IRF3, we Tris-HCl [pH 6.8], 15% glycerol) were applied on the gel and electro- inactivated the NES of IRF3 to avoid its interference. Point phoresed for 60 min at 35 mA on ice, followed by immunoblotting. mutations of the NES of IRF3, the IL139/140MM modification Data collection and statistics (designated as IRF3-NES), abolished the nuclear export function of All experiments were repeated at least three times, and data from parallel IRF3 (25). As shown in Fig. 1D, in uninfected cells, GFP-IRF3 treatments were acquired. The significance of the difference between was mainly localized in the cytoplasm, whereas GFP-IRF3(-NES) control and experimental groups was tested using an unpaired two-tailed was found in the nucleus and cytoplasm, indicating that IRF3 could Student t test. Fluorescent imaging analysis was performed in a blind enter and stay in the nucleus when the NES was deficient. We then fashion. truncated IRF3 from both the N-terminal and the C-terminal regions Results (Fig. 1C). As shown in Fig. 1D and 1E, the C-terminal truncations (aa 1–382, 1–357, and 1–201) in fusion with GFP all retained the Mapping of the NLS domain of IRF3 ability to translocate into the nucleus, whereas the N-terminal trun- Downloaded from It is well known that IRF3 is predominantly localized in the cy- cations (GFP-IRF3131-427) lost the ability to accumulate in the toplasm prior to virus infection and is imported into the nucleus nucleus. However, the N-terminal truncation GFP-IRF364-427 following infection. A previous study identified two basic amino retained the ability to translocate into the nucleus. These results 77 78 acid residues ( KR ) as the NLS of IRF3 (30). However, during indicate that the NLS domain of IRF3 resides within the N-terminal our work on the function and regulation of IRF3, we found, as region of aa 64–131.

expected, that the composition and function of IRF3 are much http://www.jimmunol.org/ more complicated than anticipated. Therefore, we started to sys- Mutational analysis of IRF3 NLS tematically characterize the nature and function of IRF3. Because most of the known NLS domains contain clusters of basic We first analyzed the subcellular distribution and the NLS do- residues, we examined the IRF3 sequence to search for clusters main of endogenous IRF3 by nucleus–cytoplasm extraction and of basic residues within the region of aa 64–131 and then per- immunofluorescence assays. As shown in Fig. 1A, prior to virus formed site-directed mutagenesis to evaluate their contribution infection, IRF3 was predominantly localized in the cytoplasm, to IRF3 nuclear accumulation. As shown in Fig. 2A, seven clusters whereas a weak band of IRF3 was detected in the nucleus; upon of basic amino acids could be recognized and were replaced by SeV infection, IRF3 began to translocate into the nucleus. The uncharged amino acids. The resulting IRF3 mutants were des- same results were obtained in the immunofluorescence assay. As ignated RK68/70SQ, KR77/78NG, R81L, RK86/87LQ, R91L, by guest on September 27, 2021

FIGURE 1. Mapping of the IRF3 NLS do- main. (A) HEK 293 cells were infected with SeV (50 HA/ml) at the indicated time before nuclear–cytoplasm extraction assays were performed; 5% of cytoplasmic extracts and 10% of nuclear extracts were subjected to SDS-PAGE analysis and immunoblotting with the indicated Abs. (B) MEFs were left unin- fected or were infected with SeV for 12 h, immunofluorescently stained with IRF3 Ab (green) and DAPI (blue), and examined using confocal microscopy. Scale bars, 10 mm. (C) Schematic presentation of IRF3-GFP trunca- tion mutants. (D) HEK 293 cells were trans- fected with the indicated IRF3-GFP truncation mutant plasmids for 24 h before confocal microscopy. Scale bars, 10 mm. (E) The in- tensities of nuclear/cytoplasmic GFP were quantitated using the ImageJ processing pro- gram and normalized to that of total GFP. 4 CHARACTERIZATION OF IRF3 BIPARTITE NLS Downloaded from

FIGURE 2. Screening of the IRF3 NLS domain for key basic amino acids. (A) Schematic presentation of IRF3 and mutation of the basic amino 2 2 acids to neutral amino acids in the region spanning aa 64–130. (B) Irf3 / http://www.jimmunol.org/ Irf72/2 MEFs were transfected with the indicated IRF3 site-directed mutant plasmids for 24 h and then left uninfected or infected with SeV for 8 h before luciferase assays and immunoblotting. The immunoblots show FIGURE 3. Determination of the key basic amino acids that specify the protein expression of IRF3 variants and endogenous GAPDH in the IRF3 nuclear import. (A) HEK 293 cells were transfected with the indi- SeV-infected group. Assays were performed in triplicate. cated IRF3 site mutant plasmids for 24 h and then left uninfected or infected with SeV for 8 h before SDS-PAGE or native-PAGE, followed by immunoblotting with anti-Flag Ab. Endogenous GAPDH was detected as RK96/98SQ, and K105Q. Upon virus infection, IRF3 undergoes a loading control. (B) HEK 293 cells were transfected with the indicated dimerization and nuclear accumulation and then activates the IFN-b

IRF3 site mutant plasmids for 24 h and then left uninfected or infected by guest on September 27, 2021 promoter. The site mutation that results in a deficiency in IRF3 with SeV for 8 h before nuclear-cytoplasm extraction assays were per- nuclear transportation fails to activate IFN-b. We tested whether the formed. Five percent of cytoplasmic extracts and 10% of nuclear extracts above IRF3 mutants could activate IFN-b by transfecting these site were subjected to SDS-PAGE analysis and immunoblotting with the in- 2 2 2 2 mutations into Irf3 / Irf7 / MEFs together with IFN-b promoter dicated Abs. Assays were performed in triplicate. LMNB1 and b-tubulin reporter plasmids. Irf32/2 Irf72/2 MEFs were used to avoid the were used as markers for nuclear and cytoplasmic proteins, respectively. interference of endogenous IRF3 and IRF7. As shown in Fig. 2B, the mutants RK68/70SQ, R91L, RK96/98SQ, and K105Q retained the ability to activate IFN-b, indicating that these are not the amino (Fig. 2B) or other cell lines, such as HeLa cells and HEC-1B cells acids that specify IRF3 nuclear accumulation. In contrast, the (Supplemental Fig. 1). Because RK86/87LQ mutation totally mutants KR77/78NG, R81L, and RK86/87LQ failed to activate abolished IRF3 nuclear import and IRF3-mediated IFN-b induc- IFN-b, indicating these basic amino acids may be responsible for tion in different cell lines, irrespective of its expression level, all of IRF3 nuclear import and act as part of the NLS. the data clearly support the role of RK86/87 in nuclear localiza- tion. We observed the same results when we transfected the syn- Determination of specific basic amino acids that contribute to thetic viral RNA analog polyinosinic-polycytidylic acid into the IRF3 nuclear import cells in place of SeV infection (data not shown). The decrease in We then determined whether the failure of the IRF3 mutants in cytoplasmic IRF3 could not be detected (Fig. 3B, lower panel), IFN-b activation lies in their nuclear inaccessibility. Because it is probably because IRF3 expression was driven by the strong CMV well known that dimerization and nuclear import are two pre- promoter, and the persistent synthesis of IRF3 may obscure the requisites for IRF3 activation (31), we first determined at which turnover of cytoplasmic IRF3. These results indicate that both step these mutants lost their activity. As shown in Fig. 3A, all of clusters of basic residues (KR77/78 and RK86/87) are essential for these mutants retained the ability to dimerize, but dimer forma- nuclear import of IRF3. tion of mutant KR77/78NG was reduced. As shown in Fig. 3B, To determine the specific contribution of these clusters of basic dimerized KR77/78NG failed to be imported into the nucleus after residues to IRF3 nuclear import, we generated NES-deficient IRF3 virus infection, indicating that basic residues KR77/78 are re- mutants (IL139/140MM), resulting in the mutants IRF3-NES, quired for IRF3 nuclear import. The mutant RK86/87LQ also KR77/78NG-NES, R81L-NES, and RK86/87LQ-NES. As shown failed to be imported into the nucleus following SeV infection, but in Fig. 4A, prior to virus infection, IRF3-NES, KR77/78NG-NES, R81L retained the ability to translocate to the nucleus (Fig. 3B). and R81L-NES were able to be imported into the nucleus; upon The expression level of RK86/87 mutant was reduced ∼35% virus infection, the nuclear localization of these mutants was compared with other IRF3 variants in HEK 293 cells (Fig. 3A). In markedly enhanced, and the nuclear IRF3 following virus infec- contrast, there was no significant difference in expression between tion was hyperphosphorylated, which could be readily recognized RK86/87LQ and other IRF3 variants in Irf32/2 Irf72/2 MEFs by the band shift in Fig. 4A. However, RK86/87LQ-NES could The Journal of Immunology 5

nucleus, even in the absence of NES function, suggesting that amino residues RK86/87 represent an essential part of the IRF3 NLS. In contrast, the mutant KR77/78NG-NES could enter the nucleus, although at low efficiency (Fig. 4A), indicating that the role of KR77/78 in IRF3 nuclear import could be partially offset by NES in intact IRF3. Although KR77/78NG-NES and R81L- NES were able to translocate into the nucleus after virus infection, they were unable to activate IFN-b, implying that these two mutants were deficient in the subsequent transcription activation, which was tested in our subsequent experiments. The same results were observed when we transfected these mutants into HEC- 1B cells (Supplemental Fig. 2), indicating that the function of these basic clusters responsible for IRF3 nuclear import is not cell type specific. Immunofluorescence analysis of IRF3 nuclear accumulation We carried out immunofluorescence assays to confirm the role of the clusters of basic residues in IRF3 nuclear import. We used a tet-

off expression system to avoid the consistent overexpression of Downloaded from transfected IRF3. IRF3 wild-type (WT), KR77/78NG, R81L, and RK86/87LQ were reintroduced into Irf32/2 Irf72/2 MEFs, and doxycycline was added to terminate the translation of IRF3. As shown in Fig. 5A, IRF3 WT and R81L were localized in the nu- cleus following SeV infection but KR77/78NG and RK86/87LQ

were not. These results were in accordance with data from the http://www.jimmunol.org/ nuclear–cytoplasm extraction assays. Furthermore, we performed the same assays with the NES-deficient mutants, including IRF3- NES, KR77/78NG-NES, R81L-NES, and RK86/87LQ-NES. As shown in Fig. 5A, in uninfected cells, IRF3-NES localized in both the nucleus and the cytoplasm, whereas KR77/78NG-NES and R81L-NES localized predominantly in the cytoplasm, and only a small amount was detected in the nucleus. In contrast, RK86/ 87LQ-NES was detected solely in the cytoplasm. Upon SeV in- fection, IRF3-NES localized exclusively in the nucleus. KR77/ by guest on September 27, 2021 78NG-NES and R81L-NES localized predominantly in the nu- cleus, but a residual amount of IRF3 could be detected in the cytoplasm. However, RK86/87LQ-NES was still retained in the cytoplasm even after virus infection. Consistent with the nuclear– FIGURE 4. Determination of the contribution of the key basic clusters. cytoplasm extraction assays, these results demonstrated that A ( ) HEK 293 cells were transfected with the indicated IRF3 site mutant KR77/78 and RK86/87 are the critical basic amino acid residues plasmids for 24 h and then left uninfected or infected with SeV for 8 h responsible for IRF3 nuclear accumulation, and they make up the before nuclear-cytoplasm extraction assays were performed. Five percent of cytoplasmic extracts and 10% of nuclear extracts were subjected to bipartite NLS of IRF3. B 2/2 SDS-PAGE analysis and immunoblotting with the indicated Abs. ( ) Irf3 Integration of the DNA-recognition motif and NLS domain Irf72/2 MEFs were transfected with the indicated IRF3 site mutant plas- mids for 24 h, and then left uninfected or infected with SeV for 8 h before The x-ray crystallographic analysis of complexes between the DNA luciferase assays. Data are mean 6 SD (n = 3). (C) HEK 293 cells were sequence that covers the PRD III–I region (nt 296 to 264) of the transfected with the indicated IRF3 site mutant plasmids for 24 h and then IFN-b enhancer and the recombinant DBD of IRF3 revealed that left uninfected or infected with SeV for 8 h before SDS-PAGE or native- the IRF3 DBD binds to DNA through positioning the a3 helix in PAGE, followed by immunoblotting with anti-Flag Ab. Assays were per- the major groove and loop L1 near the minor groove (32). As formed in triplicate. shown in Fig. 6A, K77, R78, R81, R86, and K87 are all positioned in the a3 helix, making direct or water-mediated hydrogen bonds not translocate into the nucleus, with or without virus infection with the nitrogenous bases or phosphate backbone. To determine (Fig. 4A). Furthermore, we tested the ability of the mutants to the contribution of these basic amino acids to DNA binding, we activate IFN-b. As shown in Fig. 4B, IRF3-NES activated IFN-b expressed recombinant IRF3 DBD (1–115 aa) and its site-directed production following virus infection, but it failed to activate mutants (Fig. 6B). First, we performed a dose-dependent EMSA to IFN-b before virus infection, even though it was able to translo- measure the binding ability of IRF3 DBD to the ISRE sequence. cate into the nucleus. Dimerization analysis showed that nuclear As shown in Fig. 6C, when the molar ratio of IRF3/DNA was IRF3-NES, KR77/78NG-NES, and R81L-NES were in a mono- increased, the mobility of the IRF3–DNA complex became pro- meric state before virus infection (Fig. 4C). The full-length IRF3 gressively reduced in the gel. At a ratio . 1:1, all of the DNA is monomer could not bind to the promoter ISRE and, thus, is not bound, and no dissociation of the complex occurs during elec- able to activate IFN-b without virus infection. KR77/78NG-NES, trophoresis. In contrast, recombinant IRF3 could not bind with the R81L-NES, and RK86/87LQ-NES failed to activate IFN-b, de- mutated DNA probe, indicating that the observed band shift spite the fact that all three mutants could dimerize when stimu- resulted from the specific binding of IRF3 and the probe DNA lated by virus. RK86/87LQ-NES failed to be imported into the (Fig. 6C). We observed that adding IRF3 Ab to the mixture of 6 CHARACTERIZATION OF IRF3 BIPARTITE NLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 5. Immunofluorescent staining analysis of nuclear import of IRF3 variants. (A) Irf32/2 Irf72/2 MEFs were transfected with the indi- cated IRF3 site mutant plasmids (tet-off expression system) for 24 h, treated with doxycycline (1 mg/ml), left uninfected or infected with SeV for 12 h, immunofluorescently stained with IRF3 Ab (green) and DAPI (blue), and examined using confocal microscopy. Scale bars, 10 mm. (B) The fluorescent intensities of nuclear/cytoplasmic IRF3 were quantitated using the ImageJ processing program and normalized to those of total FIGURE 6. IRF3 NLS is involved in DNA-binding activity. (A) IRF3. Schematic diagram of IRF3–DNA contacts based on the crystal structure of the complex between the DNA sequence, which covers the PRD III–I region (nt 296 to 264) of IFN-b enhancer, and isolated IRF3 DBDs. (B) recombinant protein IRF31–115 and the DNA probe severely im- The purity and quantity of recombinant IRF31–115,IRF31–115 (KR77/ paired the interaction between IRF31–115 and the DNA probe 78NG), IRF31–115 (R81L), and IRF31–115 (RK86/87LQ) were examined compared with the control IgG–treated lane, resulting in more by SDS-PAGE, followed by Coomassie blue staining. (C)EMSAwas unbound probe DNA in the IRF3 Ab–treated lane (Supplemental performed by incubating WT or mutated DNA probe (20 mM) with in-

Fig. 3) and indicating that the antigenic determinants of IRF3 creasing amounts of recombinant IRF31–115.(D) EMSA was performed by recognized by these IRF3 Abs may overlap with the region re- incubating WT or mutated DNA probe (20 mM) with recombinant IRF31–115, sponsible for DNA binding or that DNA binding may have IRF31–115 (KR77/78NG), IRF31–115 (R81L), and IRF31–115 (RK86/87LQ) changed the conformation of IRF3. Although super shift was not (each 40 mM). observed, the specific binding between recombinant IRF31–115 and the DNA probe was confirmed using the mutated probe. Next, we tested the DNA-binding ability of the IRF3 mutants at a 2:1 ratio. The antiviral function of IRF3 requires intact NLS As shown in Fig. 6D, both KR77/78NG and R81L completely To explore the potential effects of these basic amino acids re- lost their DNA-binding ability, whereas RK86/87LQ retained its sponsible for both nuclear import and DNA binding on the IRF3- DNA-binding activity but at a reduced efficiency compared with mediated antiviral response, we tested the replication capacity of that of WT IRF3. Such data indicated that the basic amino acid VSV-GFP in Irf32/2 Irf72/2 MEFs stably expressing vector and clusters responsible for IRF3 nuclear import are also critical the indicated IRF3 mutants. As shown in Fig. 7A, IRF3 WT and for DNA binding. This also may explain why KR77/78NG-NES IRF3-NES expression markedly suppressed VSV-GFP replication. R81L and R81L-NES could translocate into the nucleus but were Consistently, VSV-GFP titers were much lower in Irf32/2 Irf72/2 unable to activate IFN-b after SeV infection. These results dem- MEFs complemented with IRF3 WT or IRF3-NES than were onstrate that the two functions of IRF3, DNA binding and nuclear those in control vector–restored Irf32/2 Irf72/2 MEFs (Fig. 7B). import, are integrated in its N-terminal region. In contrast, Irf32/2 Irf72/2 MEFs restored with KR77/78NG, The Journal of Immunology 7

FIGURE 7. IRF3 NLS mutants are defective in inducing antiviral responses. (A) Irf32/2 Irf72/2 MEFs were transfected with the indicated IRF3 site mutant plasmids for 24 h and infected with VSV- GFP (multiplicity of infection [MOI] = 0.01) before microscopy. (B) The cell culture supernatant in (A) was collected to measure the VSV-GFP titer by plaque assay. (C) The cell culture supernatant in (A) was collected to measure the secretion of IFN-b cytokine. Assays were performed in triplicate. Bar graphs show mean 6 SD (n = 3). ND, not detected. Downloaded from

R81L, RK86/87LQ, or their relative NES mutants behaved as and IRF7 may be homologous to IRF5 and IRF3, respectively. Our http://www.jimmunol.org/ Irf32/2 Irf72/2 MEFs complemented with vector plasmid control. subsequent research goal is to define the NLS of IRF6 and IRF7. In line with these results, Irf32/2 Irf72/2 MEFs restored with Sequence alignment (Fig. 8B) reveals that IRFs display high IRF3 WT or IRF3-NES exhibited remarkably enhanced IFN-b overall homology among the DNA-recognition helix, which is production following SeV infection, whereas Irf32/2 Irf72/2 critical for the DNA-binding activity of the IRF family proteins. MEFs complemented with vector or other mutants showed no However, only IRF3 uses this region as an NLS domain, and the IFN-b secretion under the same conditions (Fig. 7C). Sequence key basic amino acids corresponding to IRF3 NLS are not con- alignment and structural analysis of IRF3 NLS domain revealed served in the IRF family. The unique NLS domain on IRF3 is that IRF3 NLS is highly reserved among different species but highly conserved from fish to primates (Fig. 8C). Bipartite basic unique among the IRF family proteins (Fig. 8). Collectively, the signals typically consist of interdependent basic amino acid by guest on September 27, 2021 data indicate that these basic amino acids that are responsible for clusters separated by a flexible linker of 9–12 aa (29, 36). The both nuclear import and DNA binding are highly reserved and are linker between the two basic clusters (KR77/78 and RK86/87) on essential in virus-induced IFN-b production and cellular antiviral IRF3 NLS domain contains only seven residues, shorter than the responses. length of the conventional linkers. The IRF3 NLS domain contains an a-helical motif, and KR77/78 and RK86/87 are 90˚ apart Discussion (Fig. 8D), which makes a distance of 1–2 aa for the spatial dis- Our results define a bipartite NLS that is integrated within the tance between the two basic clusters that interact with the minor DNA-recognition region of IRF3. We mapped the NLS of IRF3 to binding site and major binding site in importin-a, respectively. A aa 64–130, partially overlapping with the DBD. Basic amino acids previous report also showed that Ty1 integrase contains a bona KR77/78 and RK86/87 are required for efficient nuclear import of fide bipartite NLS with a 29-aa linker (37). Our data, together with IRF3. Significantly, we demonstrate that the NLS of IRF3 also these reports, reveal that the traditional definition of NLS is too plays an important role in the DNA-binding activity. restrictive; the linker length can vary depending on amino acid The IRF family contains nine mammalian members (IRF1, IRF2, composition and conformation. These findings also allow us to IRF3, IRF4, IRF5, IRF6, IRF7, IRF8, and IRF9), which are most expand the definition of a classic bipartite NLS consensus beyond conserved in their DBD. IRF1 and IRF2, which are closely related the previous limitations of a 9–12-aa linker. to each other, contain a conserved NLS located immediately With regard to the nature of the NLS of IRF3, there is clear C-terminal to the DBD, involving aa 120–138 (33). IRF4, IRF8, difference between our results and those of a previous study (30). and IRF9 are highly conserved with each other and use the ho- We showed in this study that IRF3 NLS is bipartite, consisting of mologous NLS (aa 66–85) to direct their accumulation in the residues KR77/78 and RK86/87, whereas the earlier work claimed nucleus (34). Interestingly, IRF5 contains two monopartite con- that the residues KR77/78 are the sole determinant of IRF3 NLS. sensus NLSs, a N-terminal NLS and a C-terminal NLS (35). Our Such a disparity could result from the use of different experi- study, together with previous reports, demonstrated that the NLSs mental methodologies. Examination of the earlier work by Kumar of IRFs are generally positioned within or in close proximity et al. (30) reveals several likely pitfalls in their studies. First, they to the DBD, and the principal type of NLSs for these IRFs is used only GFP-IRF31–131 fusion for nuclear-localization studies structurally bipartite. Sequence alignment reveals that the con- by fluorescence microscopy. Small proteins can pass the nuclear servation of NLS domain of the IRF family proteins is related to pore in an energy- and -independent manner (38); in fact, their phylogenetic relationship (Fig. 8A, 8B). The NLSs of IRF the GFP-IRF31–131 fragment (∼42 kDa) is able to diffuse between family proteins have been identified, with the exception of IRF6 the nucleus and the cytoplasm of cells (Fig. 1D), thus giving rise and IRF7. Phylogenetically, IRF6 is most closely related to IRF5, to indefinite judgment of NLS determinants. Second, they used and IRF7 is most closely related to IRF3. Thus, the NLSs of IRF6 a cell line that contains endogenous WT IRF3. Because IRF3 can 8 CHARACTERIZATION OF IRF3 BIPARTITE NLS

FIGURE 8. Sequence alignment and structural analysis of IRF3 NLS domain. (A) Phylogenetic anal- ysis of IRF family proteins. (B) Sequence alignment of Downloaded from the DNA recognition a-helix of IRF family proteins. (C) Sequence alignment of the IRF3 NLS domain from different animal species. (D) Illustration of the NLS domain of IRF3. http://www.jimmunol.org/ by guest on September 27, 2021

form dimers, the intrinsic IRF3 may influence the behaviors of structure of IRF3, and the dispelling of negative charge at RK96/98 GFP-fused IRF3 or its mutants. In our study, we characterized the appears to be equal to the C-terminal phosphorylation in unleashing IRF3 NLS through two experimental methods: nuclear–cytoplasm the autoinhibition state of IRF3. extraction assays and fluorescence microscopy. We also used Irf3/ It is widely accepted that dimerization of IRF3 is crucial for Irf7-null MEFs to avoid the interference of endogenous native nuclear import and transcriptional activity of IRF3. However, anal- IRF3. Furthermore, we also analyzed the role of the key amino ysis using the mutant IRF3-NES demonstrated that the NES-deficient acid residues in type I IFN responses and IRF3-mediated antiviral monomeric IRF3 could enter the nucleus but was unable to activate activity. All of these studies point out that the amino acid residues the IFN-b promoter, suggesting that the NLS of IRF3 is not masked KR77/78 and RK86/87 make up the bipartite NLS of IRF3 and are in the autoinhibitory state. Monomeric IRF3 in the autoinhibitory essential to virus-induced innate immunity. state cannot associate with CBP/p300 and is not ready to realign the Based on the crystal structure of IRF3 (39), inactive IRF3 is in DBD for transcriptional activity, which may account for the in- “the autoinhibitory state,” in which two regions corresponding ability of IRF3-NES to activate the IFN-b promoter prior to viral to residues 380–427 and 98–240 of IRF3 interact to form a infection. These results also suggest that dimerization is not a pre- closed structure in the inhibited state (40). Virus-induced mul- requisite for IRF3 nuclear import. tiple phosphorylation of C-terminal serine/threonine residues or It is notable that IRF proteins contain both NLSs and NESs; thus, phosphorylation-mimetic mutation of IRF3 (IRF3-5D) introduces regulation of subcellular distribution is emerging as a common massive negative charges, leading to unmasking of the NLS do- method to control IRF functions. The predominant cytoplasmic main and realignment of the DBD for transcriptional activity. localization of IRF3 before virus infection implies that nuclear Interestingly, our study showed that mutation RK96/98SQ in IRF3 export is more dominant than nuclear import prior to virus infection. generated a strong activator of IFN-b (Fig. 2B). However, IRF3 resides predominantly in the nucleus after viral The RK96/98SQ mutant alone was able to stimulate IFN-b ex- infection, indicating that nuclear import is dominant when IRF3 is pression as strongly as virus infection. These results suggest that activated. Because IRF3 is a major transcriptional factor for type I RK96/98 may be directly involved in the interaction of residues IFN responses, elaborate regulation at the steps of nuclear import 98–240 with the C-terminal phosphor-acceptor region in the closed and export can be envisaged. Therefore, further characterization of The Journal of Immunology 9 the molecular processes will help to reveal the mechanisms of IRF3 a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory ele- ments. Cell 54: 903–913. activation and IRF3-mediated antiviral immunity. 20. Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T. M. Kundig,€ R. Amakawa, K. Kishihara, A. Wakeham, et al. 1993. Targeted Acknowledgments disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75: 83–97. We thank Dr. Hong-Bing Shu for kindly providing reporter plasmids and 21. 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