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 28, 2021. Guo J Immunol 2015; 195:289-297; Prepublished online 20 May 2015; doi: 10.4049/jimmunol.1500232 http://www.jimmunol.org/content/195/1/289 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2015/05/20/jimmunol.150023 Material 2.DCSupplemental http://www.jimmunol.org/ References This article cites 40 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/195/1/289.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. 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 proteins, and nuclear trafficking is central to the function of transcription factors. IFN regulatory factor (IRF)3 is a master transcription factor 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: 289–297. 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 cytokines (1). Upon as positive regulators of IFN-a/b gene 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 genes (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 28, 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 protein 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 290 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.