Alternative Splicing Transcripts of Zebrafish LGP2 Differentially Contribute to IFN Antiviral Response

This information is current as Qi-Min Zhang, Xiang Zhao, Zhi Li, Min Wu, Jian-Fang Gui of September 27, 2021. and Yi-Bing Zhang J Immunol published online 4 December 2017 http://www.jimmunol.org/content/early/2017/12/03/jimmun ol.1701388 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 4, 2017, doi:10.4049/jimmunol.1701388 The Journal of Immunology

Alternative Splicing Transcripts of Zebrafish LGP2 Gene Differentially Contribute to IFN Antiviral Response

Qi-Min Zhang,*,† Xiang Zhao,*,† Zhi Li,* Min Wu,*,† Jian-Fang Gui,*,† and Yi-Bing Zhang*,†

In mammals, RIG-I like receptors (RLRs) RIG-I and melanoma differentiation–associated gene 5 (MDA5) sense cytosolic viral RNA, leading to IFN antiviral response; however, LGP2 exhibits controversial functions. The same happens to fish LGP2. In this study we report that three zebrafish LGP2 splicing transcripts, a full-length LGP2, and two truncating variants, LGP2v1 and LGP2v2, play distinct roles during IFN antiviral response. Overexpression of the full-length LGP2 not only potentiates IFN response through the RLR pathway, in the absence or presence of poly(I:C) at limited concentrations, but also inhibits IFN response by relative high concentrations of poly(I:C) through functional attenuation of signaling factors involved in the RLR

pathway; however, LGP2v1 and LGP2v2 only retain the inhibitory role. Consistently, LGP2 but not LGP2v1 and LGP2v2 confers Downloaded from protection on fish cells against spring viremia of carp virus (SVCV) infection and at limited expression levels, LGP2 exerts more significant protection than either RIG-I or MDA5. Further data suggest that in the early phase of SVCV infection, LGP2 functions as a positive regulator but along with SVCV replicating in cells up to a certain titer, which leads to a far more robust expression of IFN, LGP2 switches to a negative role. These in vitro results suggest an ingenious mechanism where the three zebrafish LGP2 splicing transcripts work cooperatively to shape IFN antiviral responses. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ n mammals, host cells possess an innate ability to recognize Extensive studies have suggested a canonical paradigm of RLR virus infection and mount a powerful antiviral response. Virus signaling. RIG-I undergoes a conformational change upon binding I RNA accumulating in the cytoplasm of infected cells can be to 59ppp- or 59pp-dsRNA through RD and domain, sensed through cytosolic pattern recognition including therefore releasing N-terminal CARDs from an autoinhibitory retinoic acid–inducible gene-I (RIG-I) and melanoma differentiation– state in an ATP-dependent manner, and subsequently interacting associated gene 5 (MDA5), two well-characterized members with adaptor mitochondrial antiviral signaling (MAVS) of the RIG-I like receptor (RLR) family, both of which harbor for downstream signaling cascade. MDA5 preferentially binds to three conserved domains by the presence of two N-terminal long dsRNAs to form protein-coated filaments, thus resulting in by guest on September 27, 2021 caspase activation and recruitment domains (CARDs), a central oligomerization of tandem CARDs to activate MAVS (3). The DExD/H-box RNA helicase domain, and a C-terminal regulatory resultant MAVS activation finally facilitates protein kinases domain (RD) (1, 2). TANK-binding kinase 1 (TBK1) to phosphorylate and activate IFN regulatory factors 3/7 (IRF3/7), leading to the induction of type I IFN and IFN-stimulated (ISGs) for the establishment of a broadly effective antiviral state (1, 2). Mediator of IRF3 ac- *State Key Laboratory of Freshwater Ecology and Biotechnology, Key Laboratory of Aquaculture Disease Control of Ministry of Agriculture, Institute of Hydrobiology, tivation (MITA) has also been reported as a scaffold protein for Chinese Academy of Sciences, Wuhan 430072, China; and †University of Chinese linking TBK1 and IRF3 to the MAVS complex upon RNA virus Academy of Sciences, Beijing 10049, China infection (4), although it is believed to primarily participate in ORCID: 0000-0002-7634-338X (Q.-M.Z.). virus DNA–directed IFN signaling (5). Received for publication October 3, 2017. Accepted for publication November 5, Laboratory of genetics and physiology 2 (LGP2) is the third 2017. member of the RLR family, which shares the helicase domain and This work was supported by a grant from the Strategic Priority Research Program of RD but lacks the two N-terminal CARDs that are required for the Chinese Academy of Sciences (XDA08010207), grants from the National Natural Science Foundation (31572646 and 31772875), and a grant from the Freshwater signaling (6, 7). The absence of N-terminal CARDs makes it Ecology and Biotechnology Laboratory (2016FBZ01). difficult to understand LGP2’s roles in RLR-mediated signaling, Address correspondence and reprint requests to Dr. Yi-Bing Zhang, State Key Lab- and the exact function of LGP2 is puzzling (3). LGP2 is initially oratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chi- nese Academy of Sciences, Donghu South Road 7, Wuhan 430072, China. E-mail identified as a negative regulator of IFN response triggered by address: [email protected] Sendai virus and Newcastle disease virus, which are sensed ex- The online version of this article contains supplemental material. clusively by RIG-I (7–10), and also by polyinosinic:polycytidylic Abbreviations used in this article: CARD, caspase activation and recruitment domain; acid [poly(I:C)], a synthetic dsRNA sensed by either RIG-I or ChIP, chromatin immunoprecipitation; Co-IP, coimmunoprecipitation; GCRV, grass MDA5 (9). Together with the elevated expression of LGP2 by carp reovirus; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; LGP2, labora- tory of genetics and physiology 2; LGP2v, LGP2 truncating variant; MAVS, mito- virus infection and IFN treatment, these results support a notion chondrial antiviral signaling protein; MDA5, melanoma differentiation–associated that LGP2 acts as a feedback regulator of RLR signaling (7–9). gene 5; MITA, mediator of IRF3 activation; ORF, open reading frame; poly(I:C), However, three separate strains of LGP22/2 mice exhibit disparate polyinosinic:polycytidylic acid; RD, regulatory domain; RIG-I, retinoic acid–induc- ible gene I; RLR, RIG-I like receptor; RT-qPCR, quantitative real-time PCR; SVCV, phenotypes (11–13). In-depth delineation of the former two strains spring viremia of carp virus; TBK1, TANK-binding kinase 1; TCID50, 50% tissue of deficient mice infected with a similar virus set show a negative culture–infective dose. or positive role of LGP2 in RLR signaling, respectively (11, 12). Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Analysis of the third strain reveals that LGP2 is not essential for

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701388 2 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS the induction of innate immune defenses but rather controls CD8+ Zebrafish (Danio rerio) strain AB were raised, maintained, reproduced, and T cell survival and fitness in response to Sendai virus, Dengue staged according to standard protocols. For viral infection, i.p. injection was m 8 virus type 2, or West Nile virus (the latter two are sensed by RIG-I performed using 50 lofSVCV(10TCID50 per ml) per fish. and MDA5) (13). RNA extraction, cDNA synthesis, and RT-PCR Despite these differences, the former two LGP22/2 mice show a common deficiency in resistance to encephalomyocarditis virus Total RNA was extracted using Trizol Reagent (Invitrogen), and the RNA was treated with RNase-free DNase I (Promega) according to the manu- infection (11, 12). Because encephalomyocarditis virus RNA is facturer’s protocol. The DNase I-treated RNA was reverse transcribed into associated with LGP2 as a physiological agonist of MDA5- cDNA using a first-strand cDNA synthesis kit (Promega) and kept at 220˚ dependent signaling (14), these data raise a possibility that C for semiquantitative RT-PCR or quantitative real-time PCR (RT-qPCR) LGP2 may function as a positive regulator of MDA5 signaling, analysis. For RT-qPCR, all samples were analyzed in triplicate and the expression values were normalized to b-actin. Primers used for RT-PCR which is indeed supported by in vitro assays where LGP2 ap- analysis are listed in Supplemental Table I. pears to activate MDA5-dependent signaling but repress RIG-I– dependent signaling (15, 16). Mechanistically, LGP2 facilitates Plasmids MDA5 signal transduction dependent of its RNA binding activity For coimmunoprecipitation (Co-IP) assays, expression plasmids LGP2- and ATP hydrolysis activity (16–20), and also by modulation of myc, LGP2v1-myc, and LGP2v2-myc were generated by insertion of the MDA5-RNA interaction (19). Several studies suggest that the open reading frames (ORF) of zebrafish LGP2, LGP2v1, and LGP2v2 cellular LGP2 expression level might be a key factor for the into Not IandBamH I sites of pcDNA3.1(-)-myc vector. For luciferase regulation of a switch between positive and negative roles in assays, free-tagged expression plasmids were made by cloning the ORFs of zebrafish LGP2, LGP2v1, and LGP2v2 into BamH IandNot I RLR signal transduction: at lower levels, LGP2 synergizes with sites of pcDNA3.1(+) vector. Two dominant negative mutant plasmids, Downloaded from MDA5 but not RIG-I to augment IFN signaling; at higher levels, DrRIG-I-DN and DrMDA5-DN, were made by cloning the corre- LGP2 acts as an inhibitor of RIG-I and MDA5 signaling (16, 18, sponding sequences (corresponding to 197–937 aa of DrRIG-I and 224– 19). However, greater knowledge is required to fully appreciate 997 aa of DrMDA5) into Nhe IandKpn IsitesofpcDNA3.1(+)vector. All constructs were confirmed by sequencing. Other plasmids, including how LGP2 exerts opposing effects. DrIFN-w1pro-luc, DrIFN-w3pro-luc, EPC IFNpro-luc, DrRIG-INter, To better understand the function of LGP2, we are determined DrMDA5, DrMAVS, DrMITA, DrTBK1, DrIRF3, DrIRF7, and some to characterize zebrafish LGP2’s roles in RLR-directed IFN dominant negative mutant plasmids including CaRIG-I-DN, CaMDA5- http://www.jimmunol.org/ signaling from an evolutionary perspective. The zebrafish model DN, DrMAVS-DTM, CaMITA-CT, CaTBK1-K38M, DrIRF1DN, D D is becoming useful in the study of the vertebrate innate immune DrIFRF3DN, DrIRF7DN, DrSTAT1a- C, and DrSTAT1b- Cwerede- scribed previously (27, 37, 38). response (21). All three RLR receptors, together with the downstream molecules such as MAVS, MITA, TBK1, IRF3, and Luciferase activity assays IRF7, exist in zebrafish genomes (21, 22). Although fish IFNs a b According to previous reports (26, 27, 35), cells were seeded in 24-well are not classified into IFN- / , but group I and group II IFNs plates, 6-well plates, 3.5 cm dishes, or 10 cm dishes overnight and based on cysteine numbers (23–25), and they signal through cotransfected with various constructs at a ratio of 10:10:1 (promoter-driven specific receptors that differ from mammalian type I IFN re- luciferase plasmid/expression plasmid/Renilla luciferase plasmid pRL-TK) ceptors (24), fish RIG-I and MDA-5 direct IFN expression using FuGENE HD Transfection Reagent (Promega). Empty vector by guest on September 27, 2021 pcDNA3.1 was supplemented to ensure equal amounts of the transfected through a conserved pathway (26–30), indicating that zebrafish DNA in total among wells. If necessary, the cells were transfected again possess a highly developed immune system that is remarkably with poly(I:C), infected with SVCVor GCRV, or transfected with poly(I:C) similar to that of mammals. Surprisingly, published documents followed by virus infection. At the indicated time points, the cells were have shown antithetic biological activities of fish LGP2s (27, harvested and lysed according to the Dual-Luciferase Reporter Assay 28, 31–34), an interesting and contradictory phenomenon System (Promega). Luciferase activities were measured by a Junior LB9509 luminometer (Berthold, Pforzheim, Germany) and normalized to appeared in mammals. the amounts of Renilla luciferase activities. Unless indicated, the results In the current study, we found that three zebrafish LGP2 tran- were representative of more than three independent experiments, each scripts, a full-length LGP2 and two truncating variants, LGP2 performed in triplicate. truncating variant (LGP2v)1 and LGP2v2, are derived from a single Co-IP, Western blots, and Abs LGP2 gene and differentially contribute to host IFN response. Full- length LGP2 alone has the potential to activate IFN signaling, Co-IP assays were performed to determine the levels of phosphorylated fish IRF3. Briefly, cells were extracted in NP-40 lysis buffer (Beyotime), which is particularly significant at limited expression levels of protease inhibitors, and phosphatase inhibitors (Roche) for 30 min on ice. LGP2, but inhibits IFN induction by poly(I:C), a mimic of virus Following removal of nuclei and cellular debris by centrifugation, ade- infection, predominantly at high expression levels of LGP2. quate amounts of supernatants (e.g., four-fifths of a 10 cm dish) were LGP2v1 or LGP2v2 just show a weak negative regulation in LGP2- incubated with 50 ml of the designated phosphoprotein Ab that is linked or poly(I:C)-directed IFN response. Further studies suggested that to agarose (Immunechem) for 24 h at 4˚C. This phosphoprotein Ab can precipitate all cellular phosphorylated proteins with the phosphorylated LGP2 has bilateral functions and switches its role from a positive to serine, threonine, and tyrosine. The agarose-protein complex was washed a negative regulator in the presence of poly(I:C) transfection or five times with washing buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl, during spring viremia of carp virus (SVCV) infection. The data are 1 mM DTT, 1% NP-40]. By heating the samples for 8 min at 99˚C in helpful to understand the opposing function of LGP2 in fish and SDS-PAGE protein loading buffer (13; Beyotime), the precipitated proteins were released from the immunoprecipitated pellets, separated on mammals, which is seen in independent experiment systems. SDS-PAGE, transferred to polyvinylidene fluoride membranes (Milli- pore), and analyzed by immunoblotting with crucian carp C. auratus Materials and Methods IRF3 (CaIRF3) polyclonal Ab. Cells, virus, and zebrafish CaIRF3-specific Ab and CaPKR-specific Ab were described previously (26, 35). CaIRF7 Ab was made by immunization of rabbits with the pu- Fish cell lines, including Crucian carp (Carassius auratus L.) blastula em- rified recombinant protein of DBD domain of CaIRF7. DrLGP2-specific bryonic cells (CAB), epithelioma papulosum cyprini cells (EPC), zebrafish Ab was generated by immunization of rabbits with a purified peptide liver cells (ZFL), and ZF4, were cultured as described previously (35, 36). corresponding to 192–417 aa of zebrafish LGP2. The recognition speci- SVCV and grass carp reovirus (GCRV) were propagated and titered ficity of DrLGP2 Ab and no cross-recognition between CaIRF3 Ab and according to the method of Reed and Muench, by a 50% tissue culture– CaIRF7 Ab have been verified by immunoadsorption assays according to infective dose (TCID50) assay on EPC cells and CAB cells, respectively. our previous study (26). The Journal of Immunology 3

Chromatin immunoprecipitation assays luciferase activities. As shown in Fig. 3A, EPC IFN promoter EPC or ZF4 cells cultured in 10 cm dishes were transfected with indicated was significantly activated when LGP2 was transfected at low plasmids. Then 48 h later, the transfected cells were treated for 10 min with doses (10, 50, 100 ng). Although overexpression of LGP2 at a 1% formaldehyde at room temperature, collected by cell scraper, lysed, and high dose (200 ng) did not give an obvious stimulatory effect at ultrasonicated to obtain chromatin fragments between 100 and 1000 bp in 28 h posttransfection, extending overexpression time up to 55 h size. One-tenth of cell lysates were incubated overnight at 4˚C, with CaIRF3 unexpectedly resulted in a definite increase of luciferase activi- Ab and CaIRF7 Ab, respectively, with preimmune serum, or without any Ab as two negative controls, followed by incubation for another 2 h with ties, comparable to an effect by 10 ng of LGP2 (Fig. 3A). Under Dynabeads protein A (Invitrogen) (25 ml per well) at 4˚C to form the cross- the same conditions, neither LGP2v1 nor LGP2v2 showed any linked complexes. The cross-linked complexes were added with NaCl stimulatory potential (Fig. 3A). Consistently, overexpression of (0.2 M) at 95˚C for 30 min to reverse formaldehyde cross-links, treated LGP2butnotLGP2v1andLGP2v2inEPCcellsfor55hup- with proteinase K and RNase A sequentially at 62˚C for 30 min. Finally, the immunoprecipitated DNA was extracted by the phenol-chloroform method regulated the transcription of cellular IFN genes together with and subsequently quantified using RT-qPCR. The enrichment was determined four ISGs including IRF3, IRF7, viperin, and Mx, showing a relative to input following normalization to no-Ab controls. better stimulating effect at low doses (50, 100 ng) than at a high dose (200 ng) (Fig. 3B). Results In subsequent experiments, transfection of EPC cells with LGP2 Identification of three splicing transcripts from a single at low doses (10, 50, 100 ng) for 24 h provoked a strong activation zebrafish LGP2 gene of two zebrafish IFN promoters, derived from DrIFN-w1 and Using a pair of primers to amplify the entire ORF of zebrafish LGP2 DrIFN-w3 genes (27), whereas a high dose (200 ng) gave a weak, gene from SVCV-infected zebrafish spleen, we cloned three even undetectable activation; neither LGP2v1 nor LGP2v2 alone Downloaded from transcripts that are 2040, 1728, and 1644 bp, respectively (Fig. 1A). had any stimulatory effect (Fig. 3C). Similar results were seen The zebrafish genome harbors a single LGP2 gene in when transfection assays were carried out in CAB cells (Fig. 3D). 3, which is composed of 12 exons and 11 introns (Fig. 1B), a gene These results suggest a potential of LGP2 alone rather than organization similar to that in mouse and flounder (31, 39). These LGP2v1 or LGP2v2 to activate IFN response. zebrafish transcripts appear to originate from the single LGP2 Involvement of RLR signaling in zebrafish LGP2-activated IFN gene and are generated by alternative splicing, a regulated process http://www.jimmunol.org/ response that results in three different-size proteins, LGP2 full length, LGP2v1, and LGP2v2 (Fig. 1B). LGP2 represents the full-length Our previous results have confirmed the conservation of RLR-IFN transcript, including all 12 exons and encoding a 679 aa-protein; signaling in fish by overexpression of dominant negative mutants of LGP2v1 and LGP2v2 are two shorter ones that lack the exon 9 or fish RLR signaling molecules, including MAVS, MITA, TBK1, the exons 3 and 4, encoding a 575 aa or a 547 aa protein, re- IRF3, and IRF7, to block the activation of fish IFN promoters by spectively (Fig. 1C). The full-length LGP2-encoding protein poly(I:C) and RIG-I/MDA5 (26, 27, 30, 35, 37). The same strat- contains an N-terminal DExD/H-box RNA helicase domain egies were used to investigate the detailed molecular events in- predicated with ATP-binding motif and ATPase motif, an interme- volved in zebrafish LGP2-dependent signaling. Similarly, 50 and diate HELICc domain with RNA-binding motif, and a C-terminal 100 ng of LGP2 markedly activated the promoters of DrIFN-w1 by guest on September 27, 2021 RD domain with RNA binding loop and two Zn2+-binding mo- and DrIFN-w3, which was stably abolished by cotransfection of tifs; however, LGP2v1 and LGP2v2 have an incomplete the dominant negative mutants of fish MAVS, TBK1, IRF3, or DExDc domain and an incomplete HELICc domain, respectively IRF7 (DrMAVS-DTM, CaTBK1-K38M, DrIRF3DN, DrIRF7DN) (Fig. 1D). (Fig. 4A, 4B). An obvious blockade was seen in cells that were The three transcripts were detectable in resting cultured zebrafish cotransfected with 50 ng of LGP2 and the dominant negative cells (ZFL), and were significantly upregulated by poly(I:C) mutant of fish MITA (CaMITA-CT), although a slight, or no, re- transfection showing different expression patterns. The full-length duction in both zebrafish IFN promoter activities was detected LGP2 was most abundant and similar to Mxb, a typical ISG, was when 100 ng of LGP2 was used (Fig. 4A, 4B). upregulated during the whole inducing course, whereas the ex- In addition, the activation of EPC IFN promoter by LGP2 re- pression of LGP2v1 and LGP2v2 peaked at 6 h posttransfection quired MAVS, MITA, and TBK1, as evidenced by the results that then dropped slightly thereafter (Fig. 2A). Using a pair of universal the activation was severely inhibited by overexpression of primers to amplify a shared sequence, RT-qPCR analysis showed DrMAVS-DTM, CaMITA-CT, or CaTBK1-K38M (Fig. 4C). that total LGP2 was constitutively expressed in all zebrafish tis- LGP2 alone also provoked ISRE-containing promoter-driven lu- sues detected (Fig. 2B) and upregulated in response to SVCV ciferase activities, which was diminished by transfection of either infection, displaying a similar expression pattern to Mxb or both of zebrafish STAT1a-DC and STAT1b-DC, two dominant (Fig. 2C). Similar to that in ZFL cells, the three splicing forms negative mutants of STAT1 (37) (Fig. 4D). These results indicate were constitutively detected in immune tissues including gill, that zebrafish LGP2 induces ISG expression through the Stat1 liver, spleen, head kidney, and body kidney, and were significantly pathway. Consistently, overexpression of LGP2 in EPC cells in- induced by SVCV infection, with stronger expression of LGP2 duced an increase in IRF3 and PKR proteins (Fig. 4E), which were than of LGP2v1 and LGP2v2 (Fig. 2D). Expression comparison markedly inhibited by cotransfection with DrMAVS-DTM, showed a very low transcription level of LGP2, RIG-I, and MDA5 CaTBK1-K38M, DrIRF3DN, and DrIRF7DN, respectively (Fig. 4F). in resting tissues but an upregulated expression in SVCV-infected These results indicate that LGP2 activates IFN response by upregu- tissues with RIG-I . LGP2 . MDA5 (Fig. 2E). lation of IFN and ISG expression via a signaling pathway similar to RIG-I/MDA5 signaling. Activation of IFN response by LGP2 but not LGP2v1 and To strengthen our findings, chromatin immunoprecipitation LGP2v2 (ChIP) assays were used to compare the binding affinity of IRF3/7 To determine the roles of three zebrafish LGP2 transcript variants to IFN promoters under different conditions. As shown in Fig. 4G, in IFN antiviral response, EPC cells were transfected with dif- the binding of the endogenous IRF3 and IRF7 to DrIFN-w1 and ferent LGP2 expression constructs together with EPC IFN DrIFN-w3 promoters or endogenous EPC IFN promoter was en- promoter-driven luciferase constructs followed by detection of hanced in EPC cells transfected with LGP2 compared with cells 4 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/

FIGURE 1. Identification of three splicing transcripts of zebrafish LGP2 gene. (A) Identification of LGP2, LGP2v1, and LGP2v2 from SVCV-infected zebrafish spleen by the same pair of primers to amplify their complete ORFs. (B) Schematic diagrams of exon-intron arrangements of zebrafish LGP2 gene and generation of three LGP2 transcripts by exon skipping. The filled boxes and the thick lines indicate the exons that are differentially selected to assemble by guest on September 27, 2021 three splicing transcripts. (C) Schematic structure of LGP2, LGP2v1, and LGP2v2 proteins showing the domain differences among them. The number indicates the amino acid position. (D) Amino acid sequence alignments of zebrafish LGP2, LGP2v1, and LGP2v2 showing the localization of domains including DExDc, HELICc, and RD. transfected with an empty vector; however, the enhanced binding (P), matrix protein (M), glycoprotein (G) and RNA polymerase (L) was significantly diminished by cotransfection of TBK1-K38M, (40). RT-qPCR showed that compared with transfection of empty MAVS-DTM, and MITA-CT, respectively (Fig. 4G). Co-IP assays vector, LGP2v1 or LGPv2, overexpression of LGP2 decreased the showed that LGP2, but not LGP2v1 or LGP2v2, significantly transcription expression of SVCV genes L, N and G (Fig. 5B) but promoted the phosphorylation of IRF3 (Fig. 4H). These results upregulated the expression of cellular IFN and three representative indicate that zebrafish LGP2 activates IFN response dependent of ISGs, viperin, Mx, and IRF7 (Fig. 5C). Consistently, transfection IRF3 phosphorylation and IRF3/7 binding to IFN promoters. of LGP2 (100 ng) resulted in an over 3-fold reduction of virus titer relative to the control cells that was transfected with an empty Inhibition of virus replication by LGP2 but not LGP2v1 and vector, LGP2v1, or LGP2v2 (100 ng); this inhibitory effect was LGP2v2 through induction of IFN and ISGs comparable to that by transfection of the same amount of MDA5 To determine a possible role of LGP2, LGP2v1, and LGP2v2 in and RIG-INter (2.4- and 3.4-fold reduction versus control, re- response to virus infection, EPC cells were transfected with EPC spectively), but less than that by transfection of poly(I:C) (100 ng/ IFN luciferase construct and diverse LGP2 constructs followed by ml) (18-fold reduction versus control) (Fig. 5D). Moreover, LGP2 SVCV infection. Luciferase assays showed significant activation of exhibited a better stimulatory potential to zebrafish IFN promoters EPC IFN promoter by LGP2, with a better effect at low amounts at low doses (,100 ng) but a poorer one at high doses (.100 ng) (10, 50, 100 ng) than large (200 ng) (Fig. 5A), but not by LGP2v1 than MDA5 or RIG-INter at the same doses (Fig. 5E). These data and LGP2v2 at any an amount followed (or not) by SVCV in- indicate that zebrafish LGP2 rather than LGP2v1 and LGP2v2 3 4 fection (Supplemental Fig. 1). SVCV alone (1 10 TCID50 per confers protection on EPC cells against SVCV infection by in- ml) failed to induce an obvious activation until 48 h postinfection, duction of IFN response, and at relatively low expression levels, and following SVCV infection did not give an enhanced activation LGP2 alone exerts more significant protection than either RIG-I or of IFN promoter by LGP2, except for an additional effect MDA5. (Fig. 5A). Subsequently, the expression of SVCV genes as well as cellular Negative regulation of poly(I:C)-triggered IFN response by IFN and some ISGs was detected to evaluate the antiviral effects of three zebrafish LGP2 splicing forms LGP2, LGP2v1, and LGP2v2 against SVCV infection. The SVCV Several published studies revealed a negative role of fish LGP2 (26, genome contains five ORFs for nucleoprotein (N), phosphoprotein 32, 33), which is contradictory to the results described above. To The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Expression analysis of LGP2, LGP2v1, and LGP2v2 in ZFL cells and zebrafish tissues. (A) Semiquantitative RT-PCR analysis of the mRNAs of LGP2, LGP2v1, and LGP2v2 in ZFL by the same pair of primers. ZFL cells seeded in 3.5 cm dishes were transfected with poly(I:C) (2 mg/ml). (B and C) RT-qPCR analysis of total zebrafish LGP2 transcripts in healthy or infected zebrafish by a pair of universal primers to amplify a shared sequence of three 8 transcripts. The indicated tissues were collected from healthy zebrafish (B) or from PBS or SVCV i.p. injected (50 mlof10TCID50 per ml) zebrafish (C). (D) Semiquantitative RT-PCR analysis of the mRNAs of LGP2, LGP2v1, and LGP2v2 in five immune tissues, including gill, liver, spleen, head kidney, and 8 body kidney from zebrafish injected i.p. with SVCV (50 mlof10TCID50 per ml). (E) RT-qPCR analysis of LGP2, MDA5, and RIG-I transcripts in zebrafish tissues shown in (D). The forward and reverse primers for LGP2 mRNA amplification were designed based on the sequences that are omitted for LGP2v1 or LGP2v2 due to alternative splicing. For RT-qPCR, the expression value was expressed as relative to the corresponding expression level of b-actin (B and E) or as fold induction relative to that in corresponding PBS i.p. injected tissues, which was set to 1 (C). Error bars represent SD obtained by measuring each sample in triplicate. clarify this, similar experiments were initially performed by doses (100 or 200 ng) of LGP2v1 and LGP2v2 also displayed a transfection of EPC cells with three LGP2 expression constructs slight inhibition to DrIFN-w1 promoter under the same conditions followed by transfection of poly(I:C), an effective IFN stimulator. (Fig. 6B, 6C). Similarly, the three zebrafish LGP2 isoforms im- LGP2 alone but not LGP2v1 or LGP2v2, as expected, displayed a peded MAVS-, MITA- and TBK1-mediated IFN signaling, with a stably positive role in activating DrIFN-w1 and DrIFN-w3 pro- more significant effect by LGP2 than that by LGP2v1 and moters, and transfection of poly(I:C) (1 mg/ml) alone stimulated LGP2v2, but did not block IRF3- and IRF7-mediated signaling nearly 7-fold more activity of fish IFN promoters than 200 ng of (Fig. 6D). Interestingly, LGP2 significantly facilitated IRF7 to LGP2; however, transfection of EPC cells with LGP2 and sub- activate DrIFN-w1 and DrIFN-w3 promoters (Fig. 6D). Therefore, sequently with poly(I:C) resulted in IFN promoter-driven lucif- three zebrafish LGP2 isoforms negatively regulate poly(I:C)- erase activities significantly decreased by 23 ∼ 27% relative to triggered fish IFN response to different degrees. poly(I:C) alone, indicating a negative role of LGP2 in poly(I:C)- The inhibitory role of LGP2 was further determined by tran- triggered IFN response (Fig. 6A). scription detection of ISGs. Semiquantitative RT-PCR showed that Titration experiments showed a dose-dependent inhibition of transfection of poly(I:C) in ZFL cells strikingly induced, in a time- LGP2 in the activation of both fish IFN promoters by poly(I:C) and dependent fashion, the transcription of cellular genes involved in by RIG-I or MDA5, respectively (Fig. 6B, 6C). Intriguingly, larger IFN antiviral response, including MDA5, MAVS, IFN-w1, IFN-w3, 6 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/

FIGURE 3. Activation of IFN response by LGP2, but not LGP2v1 and LGP2v2. (A) Luciferase assay analysis of the activation of EPC IFN promoter by zebrafish LGP2, LGP2v1, and LGP2v2. EPC cells seeded in 24-well plates were cotransfected with EPC IFN promoter-driven luciferase plasmid (EPC IFNpro- luc, 200 ng), Renilla luciferase plasmid (pRL-TK, 20 ng), and each of three zebrafish LGP2 splicing variant plasmids at indicated amounts (10, 50, 100, 200 ng). At 28 and 55 h posttransfection, the cells were harvested for detection of luciferase activity. (B) RT-qPCR analysis of cellular IFN and ISGs in EPC cells transfected with LGP2, LGP2v1, or LGP2v2. EPC cells seeded in 24-well plates were transfected as in (A), with different doses of LGP2, LGP2v1, and LGP2v2 by guest on September 27, 2021 for 55 h, and then sampled for transcription detection of cellular IFN, IRF3, IRF7, viperin, and Mx. The expression value is expressed as fold inductionrelative to that in mock-transfected cells following normalization to the expression of b-actin. (C and D) Luciferase assay analysis of zebrafish IFN-w1andIFN-w3 promoters by LGP2, LGP2v1, and LGP2v2. EPC cells (C)andCABcells(D) were transfected as in (A) using DrIFN-w1pro-luc or DrIFN-w3pro-luc instead of EPC IFNpro-luc. Then 24 h later, the cells were harvested for detection of luciferase activity. *p , 0.05, **p , 0.01, Student t test.

IRF3, and Mxb; however, overexpression of LGP2 decreased the transfection of poly(I:C) (100 ng/ml) alone showed the best acti- transcription induction of these genes by poly(I:C) (Fig. 7A). vation on DrIFN-w1 and DrIFN-w3 promoters, being 4.3 ∼ 5.4- These results were further verified by RT-qPCR analysis fold or 3 ∼ 8.4-fold higher than transfection of 100 ng of LGP2 4 (Supplemental Fig. 2). In addition, poly(I:C) transfection induced alone or infection of SVCV (5 3 10 TCID50 per ml) alone. a time-dependent upregulation of LGP2, IRF3, and PKR proteins However, cotransfection of LGP2 and poly(I:C) resulted in ∼2-fold in ZFL, but overexpression of LGP2 abrogated poly(I:C)-induced reduction of IFN promoter activation relative to the transfection protein levels of IRF3 and PKR (Fig. 7B). of poly(I:C) alone (Fig. 8A). The LGP2-mediated suppression In final experiments, Co-IP assays showed that IRF3 phosphory- was augmented along with the plasmid amount increasing up to lation was enhanced in ZFL cells transfected with either LGP2 or poly 200 ng. Interestingly, the following SVCV infection did not alter (I:C) alone; however, cotransfection of LGP2 and poly(I:C) resulted the level, or only caused one additional increase, of IFN pro- in a decreased phosphorylation of IRF3 relative to transfection of poly moter activation by poly(I:C) and LGP2 individually and col- (I:C) alone (Fig. 7C). Consistently, transfection of either LGP2 or lectively (Fig. 8A). poly(I:C) alone in ZF4 cells increased the binding affinity of en- In subsequent experiments, a time-dependent inhibition of LGP2 dogenous IRF3 and IRF7 to both zebrafish IFN promoters, but on poly(I:C)-induced IFN response was shown by luciferase assays compared with transfection of poly(I:C) alone, cotransfection of using EPC IFN promoters instead of zebrafish IFN promoters LGP2 and poly(I:C) attenuated the binding of IRF3 to fish IFN (Fig. 8B). Simultaneous detection of virus titers from the super- promoters (Fig. 7D). Interestingly, poly(I:C)-mediated IRF7 binding natants of virally infected EPC cells revealed that transfection of was not diminished but enhanced by overexpression of LGP2, in- poly(I:C) alone had the best inhibitory effect on SVCV replica- dicating distinct roles of IRF3 and IRF7 in the negative regulation of tion, leading to a 5.6-, 42.2-, and 31.6-fold reduction of virus titers LGP2 on poly(I:C) signaling. relative to control cells at 12, 24, and 36 h postinfection, re- spectively, but 3.2-, 13.3-, and 17.8-fold reduction was observed Antithetic effects of LGP2 on SVCV replication in the absence for transfection of LGP2 and poly(I:C) collectively, indicating a or presence of poly(I:C) negative role of LGP2 (Fig. 8C). In this case, transfection of LGP2 To determine the antiviral effects of LGP2 in EPC cells in the alone resulted in 1.8-, 5.6-, and 4.2-fold reduction on SVCV titers absence or presence of poly(I:C), in initial experiments, the relative to control cells (Fig. 8C). These results suggest that LGP2 The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Requirement of RLR signaling molecules in LGP2-activated IFN response. (A–D) Zebrafish LGP2-directed activation of fish IFN promoters was inhibited by overexpression of dominant negative mutants of RLR signaling molecules. EPC cells were cotransfected with DrIFN-w1pro-luc or DrIFN- w3pro-luc [200 ng, (A and B)] or EPC IFNpro-luc [200 ng, (C and D)], the indicated amounts of LGP2 together with each of the dominant negative plasmids (200 ng) including DrMAVS-DTM, CaMITA-CT, CaTBK1-K38M, DrIRF1DN, DrIRF3DN, and DrIRF7DN. At 48 h posttransfection, the cells were collected for luciferase assays. (E and F) Western blot analyses of IRF3 and PKR induction by LGP2. EPC cells seeded in 24-well plates were transfected with LGP2 (50, 100, 200 ng) (E), or 50 ng of LGP2 together with the indicated dominant negative mutants of RLR signaling molecules (200 ng) (F). After 48 h, Western blotting was used to analyze the expression of IRF3 and PKR proteins using corresponding Abs. (G) ChIP analysis of the binding of IRF3 and IRF7 to fish IFN promoters in EPC cells. EPC cells seeded in 10 cm dishes were cotransfected with LGP2 (1 mg), DrIFN-w1pro-luc, or DrIFN-w3pro-luc (2.5 mg), together with each of dominant negative mutant plasmids (4 mg) including CaTBK1-K38M, DrMAVS-DTM, CaMITA-CT, or empty vector pcDNA3.1 as control. Cell lysates were immunoprecipitated with anti–IRF3-agarose beads, anti–IRF7-agarose beads, control serum-agarose beads, and no Ab-agarose beads, respectively. ChIP-derived DNA (DrIFN-w1 promoter, DrIFN-w3 promoter, and endogenous EPC IFN promoter) was quantified using RT-qPCR analysis, and the enrichment was determined relative to input following normalization to no-Ab controls. (H) Enhanced phosphorylation of IRF3/7 by zebrafish LGP2, not LGP2v1 and LGP2v2. EPC cells seeded in 10 cm dishes were transfected with LGP2, LGP2v1, or LGP2v2 (2 mg), respectively. Empty vector pcDNA3.1 was transfected in parallel as control. After 48 h, cell lysates were immunoprecipitated with the designated phosphoprotein Ab that is lined to agarose, followed by Western blot analysis of the immunoprecipitates with CaIRF3 Ab. The overexpression of LGP2, LGP2v1, and LGP2v2 was verified by Western blotting with LGP2 Ab. *p , 0.05, **p , 0.01, Student t test. 8 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/

A FIGURE 5. Inhibition of virus replication by LGP2 but not LGP2v1 and LGP2v2 through induction of IFN and ISGs. ( ) Luciferase assay analysis of the by guest on September 27, 2021 activation of DrIFN-w1 and DrIFN-w3 promoters by LGP2 followed by SVCV infection. EPC cells seeded in 24-well plates were transfected with DrIFN- 4 w1pro-luc or DrIFN-w3pro-luc (200 ng), the indicated amounts of LGP2 for 24 h, followed by infection with SVCV (10 TCID50 per ml). At 12, 24, 48 h post–SVCV infection, the cells were collected for luciferase assays. (B and C) Inhibition of virus replication by zebrafish LGP2 but not LGP2v1 and 4 LGP2v2 through induction of IFN and ISGs. EPC cells were treated as in (A). At 48 h post–SVCV infection (10 TCID50 per ml), the cells were sampled for transcription detection of SVCV genes (B) and cellular IFN and ISGs (C) by RT-qPCR. (D) Inhibition of SVCV replication by LGP2, LGP2v1, LGP2v2, MDA5, RIG-INter, and poly(I:C). EPC cells seeded in 24-well plates were transfected with poly(I:C) (100 ng/ml) or the indicated expression plasmids 4 (100 ng). Then 24 h later, the transfected cells were infected with SVCV (10 TCID50 per ml) for another 48 h. The culture supernatants were collected for detection of virus titers according to the method of Reed and Muench. The virus titer was expressed as 50% tissue culture infectious dose (TCID50 per ml). A representative of two experiments was shown. (E) Stimulatory comparison of LGP2, RIG-INter, and MDA5 on the activation of fish IFN promoters. EPC cells seeded in 24-well plates were transfected with the indicated plasmids. At 48 h posttransfection, the cells were collected for luciferase assays. *p , 0.05, **p , 0.01, Student t test. alone inhibits virus replication by induction of IFN response but in (Fig. 9B, upper panel). Under the same conditions, overexpression the presence of poly(I:C), it attenuated the inhibition of poly(I:C) of LGP2v1 or LGP2v2 with 2.5 ng had no effect on the activation on SVCV replication likely through restraining the IFN response. of both fish promoters (Fig. 9A, middle and lower panels); how- ever, overexpression of each with 200 ng resulted in an inhibition Function switch of LGP2 from positive to negative regulation of DrIFN-w1 promoter activation rather than DrIFN-w3 promoter during poly(I:C) transfection activation (Fig. 9B, middle and lower panels). A continuous titering experiment was carried out to determine the Subsequently, titration of LGP2 showed that although LGP2 reasons for the antithetic roles of zebrafish LGP2 involved in IFN alone at low amounts (10, 50, 100 ng) exhibited higher stimulatory antiviral response. Titration of poly(I:C) from 0.5 to 80 ng/ml potential than at a high amount (200 ng), no amount made a revealed that 2.5 ng of LGP2 did not inhibit or enhance the ac- difference on the activation of two zebrafish IFN promoters by 2.5 tivation of DrIFN-w1 promoter by low concentrations of poly(I:C) ng/ml of poly(I:C), but it significantly inhibited the activation by (,10 ng/ml), whereas it stably inhibited DrIFN-w1 promoter ac- 200 ng/ml of poly(I:C) (Fig. 9C). Similar results were seen for tivation by high concentrations of poly(I:C) (.10 ng/ml) EPC IFN promoter activation by titration of poly(I:C), showing (Fig. 9A). Regarding DrIFN-w3 promoter activation, 2.5 ng of that LGP2 preferentially inhibited IFN promoter activation by LGP2 did not make any difference (Fig. 9A, upper panels). high concentrations of poly(I:C) in a dose-dependent manner Compared to 2.5 ng of LGP2, 200 ng of LGP2 caused a strong (Fig. 9D, left panel). In this experiment, the construct LGP2-myc, inhibition on both zebrafish IFN promoter activation by high tagged with myc at the C terminus, lost the stimulatory potential concentrations of poly(I:C) (.8 ng/ml), whereas low concentra- on IFN promoter activation, but still retained the ability of im- tions of poly(I:C)-mediated signaling were still not influenced peding poly(I:C)-triggered IFN signaling (Fig. 9D, right panel). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Negative regulation of LGP2 on IFN signaling by poly(I:C). (A and B) LGP2-mediated inhibition on poly(I:C) signaling. EPC cells seeded in 24-well plates were transfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc, together with LGP2, LGP2v1, or LGP2v2 (200 ng each) (A), or together with the increasing amounts of LGP2, LGP2v1, or LGP2v2 (B) for 24 h, followed by transfection again with poly(I:C) (1 mg/ml). Then 24 h later, the transfected cells were collected for luciferase assays. (C and D) LGP2 inhibits poly(I:C) signaling through functional blockade of important signaling factors. EPC cells seeded in 24-well plates were transfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc (200 ng), together with the increasing amounts of LGP2, LGP2v1, or LGP2v2, and 200 ng of RIG-IN or MDA5 (200 ng) for 48 h (C), or together with 200 ng of LGP2, LGP2v1, or LGP2v2, and each of signaling molecules including MAVS, MITA, TBK1, IRF3, and IRF7 (200 ng) for 48 h (D). *p , 0.05, **p , 0.01, Student t test.

These results indicate that the exact concentration of poly(I:C) is inhibitory effect (Fig. 10A, left panel). Regarding DrIFN-w3 responsible for the positive or negative role of zebrafish LGP2. promoter, the inhibition did not happen, whereas a robust activa- tion was induced by infection of SVCV at high titers (11 ∼ 12.2- Function switch of LGP2 from positive to negative regulation 4 5 fold increase for 5 3 10 TCID50 per ml or 1 3 10 TCID50 during SVCV infection per ml) (Fig. 10A, right panel). Continuous titering experiments were performed in EPC cells A time-course analysis of SVCV infection showed a time- transfected with different doses of LGP2 followed by infection with dependent induction of two zebrafish IFN promoter activations different titers of SVCV. As shown in Fig. 10A, infection of SVCV by SVCV alone (Fig. 10B). At 36 h postinfection, overexpression alone induced a titer-dependent activation of two zebrafish IFN of 100 and 200 ng of LGP2 significantly blocked the activation of promoters. Regarding DrIFN-w1 promoter, low titers of SVCV DrIFN-w1 promoter by a high titer of SVCV infection (1 3 105 4 alone (,5 3 10 TCID50 per ml) induced a weak activation (2 ∼ TCID50 per ml) (nearly 45 and 49% reduction, respectively), but 3 7.8-fold increase versus control), comparable to that by transfec- not by low titers of SVCV infection (1 3 10 TCID50 per ml); tion of LGP2 alone (4 ∼ 10-fold increase versus control), and an however, this blockade did not happen at the early phase of SVCV additional activation was detected in LGP2-overexpressed cells infection (12 and 24 h postinfection) and not for low doses of followed by SVCV infection. High titers of SVCV alone (5 3 104 LGP2 (50 ng) at any time points post–viral infection (Fig. 10B). In 5 TCID50 per ml or 1 3 10 TCID50 per ml) stimulated a relatively another experiment, overexpression of 100 ng of LGP2 signifi- strong DrIFN-w1 promoter activity (15.9- and 16.8-fold increase cantly impeded the activation of DrIFN-w1 promoter by SVCV of 6 versus control) and, surprisingly, overexpression of 200 ng of 5 3 10 TCID50 per ml (∼35% reduction) but not by GCRVof 5 3 6 LGP2 resulted in an attenuated promoter activation (8.3- and 8.6- 10 TCID50 per ml, and no obvious inhibition was detected by fold increase versus control), by an almost 50% reduction. How- transfection of LGP2v1 and LGP2v2 at the same amount (100 ng) ever, overexpression of 50 or 100 ng of LGP2 did not cause an (Fig. 10C). Luciferase assays showed that overexpression of the 10 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/

FIGURE 7. LGP2 downregulates the expression of IFN and ISGs that are induced by poly(I:C) signaling. (A) Semiquantitative PCR analysis of poly(I:C)- induced in ZFL in the absence or presence of LGP2. ZFL cells seeded in 3.5 cm dishes were transfected with 800 ng of LGP2 or pcDNA3.1 as control. Then 2 h later, the cells were transfected again with poly(I:C) (2 mg/ml). (B) Western blot analysis of poly(I:C)-induced protein expression in by guest on September 27, 2021 ZFL in the absence or presence of LGP2. ZFL cells seeded in six-well plates were transfected as in (A). (C) LGP2 attenuates IRF3 phosphorylation, which is activated by poly(I:C) signaling. ZFL cells seeded in 10 cm dishes were transfected with LGP2 (5 mg). Then 24 h later, the cells were transfected again with poly(I:C) (1.5 mg/ml) for another 24 h, then collected for Co-IP assays with the designated phosphoprotein Ab linked to agarose. The immuno- precipitates were analyzed by Western blotting with CaIRF3 Ab. (D) LGP2 decreases the binding of IRF3/7 to fish IFN promoters that are activated by poly (I:C) signaling. ZF4 cells seeded in 10 cm dishes were cotransfected with 2.5 or 5 mg of LGP2. Then 24 h later, the cells were transfected again with or without poly(I:C) (400 ng/ml) for another 36 h. Cell lysates were immunoprecipitated with anti–IRF3-agarose beads, anti–IRF7-agarose beads, control serum-agarose beads, and no Ab-agarose beads, respectively. The precipitated DNA was used as a template to check the binding of IRF3/7 to endogenous IFN-w1/3 promoters by RT-qPCR. The enrichment was determined relative to input following normalization to no-Ab controls. *p , 0.05, Student t test. dominant negative mutant of MDA5 (MDA5-DN) attenuated tration (4 ng/ml) of poly(I:C) was transfected, because at this dose SVCV and GCRV-induced activation of DrIFN-w1 promoters, and poly(I:C) had a less stimulatory effect than LGP2 (Fig. 11B). overexpression of MDA5-DN and RIG-I–DN together resulted in However, when 1 mg/ml of poly(I:C) was used to transfect cells, much profound inhibition, indicating that SVCV and GCRV acti- which showed a better stimulatory potential than any a dose of vated an IFN response through RLR signaling (Fig. 10D). These LGP2 alone, overexpression of LGP2 significantly blocked poly(I:C)- results indicate that similar to high concentrations of poly(I:C), high induced IFN promoter activation in a dose-dependent manner. titers of SVCV-mediated signaling were easily inhibited by LGP2, Cotransfection of either LGP2v1 or LGPv2 did not make an ad- with a much stronger inhibitory effect from high amounts of LGP2. ditional inhibition, except when they were transfected at large amounts (200 ng) (Fig. 11C). In another experiment by transfec- Differential regulation of IFN response by three zebrafish tion of three LGP2 splicing forms at a high dose (200 ng) and poly LGP2 splicing forms individually and collectively (I:C) at a high concentration (1 mg/ml), LGP2 exhibited a strong Continuous titering experiments were next used to determine the inhibitory effect on two zebrafish IFN promoter activations by combined roles of three LGP2 isoforms in IFN antiviral response. poly(I:C), and LGP2v1 and LGP2v2 displayed a weak one. In the absence of poly(I:C), overexpression of LGP2v1 or LGPv2 Moreover, nearly no additional inhibition was observed when both stably blocked LGP2-mediated activation of DrIFN-w1 promoter were expressed together (Fig. 11D). These results indicate that in a dose-dependent manner, but resulted in a compromised ac- LGP2v1 and LGP2v2 have an ability to block the IFN response tivation of DrIFN-w3 promoter at large amounts (Fig. 11A). The induced by LGP2 alone, but do not help it make a more severe strongest inhibition generally happened when low amounts of inhibition on poly(I:C)-mediated IFN signaling. LGP2 were transfected, showing a best stimulatory potential, and when two variants were expressed at high levels, displaying the Discussion most robust inhibitory effect (Fig. 11A). The function relationship In this study, we identify three zebrafish LGP2 splicing transcripts of three LGP2 splicing forms was the same when a low concen- from a single LGP2 gene. They are differentially expressed in The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 8. Antithetic effects of LGP2 on SVCV replication in the absence or presence of poly(I:C). (A) Comparison of fish promoter activation by LGP2, poly(I:C), and SVCV individually and collectively. EPC cells seeded in 24-well plates were transfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc

(200 ng), with or without different doses of LGP2 (100 or 200 ng). Then 24 h later, the cells were transfected again with or without poly(I:C) (100 ng/ml) by guest on September 27, 2021 4 for another 24 h, followed by infection with or without SVCV (5 3 10 TCID50 per ml) for 28 h, and finally collected for luciferase assays. (B and C) Time- course analysis of fish promoter activation and SVCV replication by LGP2 and poly(I:C) individually and collectively. EPC cells seeded in 24-well plates 4 were transfected as in (A) by LGP2 (100 ng) and poly(I:C) (100 ng/ml) individually or collectively, followed by infection with SVCV (5 3 10 TCID50 per ml) for the indicated time points. The cells were collected for luciferase assays (B) and the culture supernatants were submitted for detection of viral titers (C). resting cells and upregulated in zebrafish tissues infected with in cells transfected with poly(I:C) at large concentrations (Figs. 3, SVCV. Among them, full-length LGP2 is the most abundant in the 6), which is subsequently verified by comprehensive analyses of infected tissues, but at the early time of infection or in certain gene expression, signaling transduction, and antiviral effects resting tissues, (e.g., gill, spleen), LGP2v1 mRNA appears to be the (Figs. 3–8). Moreover, when fish cells are transfected with poly(I:C) most abundant (Fig. 2), indicating their differential roles in host at limited concentrations, zebrafish LGP2 is still a potent stimu- immune response. Actually, full-length LGP2 not only has the lator of IFN signaling (Fig. 9). These results indicate that zebrafish potential to activate IFN antiviral response but also to block poly LGP2 is actually a bifunctional protein and might play opposing (I:C)- and SVCV-triggered IFN signaling under given conditions; functions under different conditions. Notably, zebrafish LGP2 however, the two shorter splicing variants LGP2v1 and LGP2v2 exerts its positive function not in a dose-dependent fashion, be- lose their immunoactive properties but retain an inhibitory role in cause the most stimulating potential of LGP2 is observed at low RLR signaling. Accordingly, the three zebrafish LGP2 isoforms levels rather than at high levels (Fig. 3). On the contrary, poly(I:C) differentially contribute to IFN antiviral response. stimulates IFN expression dependent of its concentration (25, 26) The data presented in this study may solve the puzzle of the (Fig. 9). When the poly(I:C) concentration reaches a certain point, controversial function of fish LGP2. Most studies have suggested a which results in much more IFN expression than zebrafish LGP2 positive role of fish LGP2 by ectopic expression of LGP2 in fish alone, the functional role of zebrafish LGP2 is switched from being cells followed by virus infection (28, 31, 41, 42). However, our a positive regulator to a negative one (Fig. 9). Thus, the stimulatory previous results have found that overexpression of crucian carp potential of cytosolic dsRNA, closely related to its concentration in LGP2 or its RD domain significantly blocks the activation of fish cells, is crucial for the determination of zebrafish LGP2’s function IFN promoter by cytosolic poly(I:C) or by RIG-I and MDA5, in a positive or negative role. respectively (27). In the above-mentioned studies, fish LGP2 is This notion is further verified by overexpression of LGP2 in fish expressed from expression vectors; therefore, the expression level cells followed by infection with SVCV, a negative-stranded RNA of LGP2 is high, as is the transfected poly(I:C) (27, 28, 31–33, 41, virus (40). Similar to the transfected poly(I:C) (27, 30), SVCV 42). In the current study, we can replicate the positive role of the infection stimulates IFN response through both RIG-I and MDA5 full-length LGP2 in the absence of poly(I:C) and the negative one (Fig. 10D). The activation of fish IFN promoters by high titers of 12 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 9. Function switch of LGP2 from positive to negative regulation during poly(I:C) transfection. (A and B) EPC cells seeded in 24-well plates were transfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc (200 ng), together with LGP2, LGP2v1, or LGP2v2 at 2.5 ng (A) or at 200 ng (B). Then 24 h later, the cells were transfected again with poly(I:C) at increasing concentrations (from 0.5 to 80 ng/ml) for another 24 h, and finally collected for luciferase assays. (C and D) EPC cells seeded in 24-well plates were transfected as in (A), by different doses of LGP2 (10, 50, 100, 200 ng) for 24 h, followed by transfection with poly(I:C) (2 or 100 ng/ml) (C), or EPC cells were transfected with 100 or 200 ng of LGP2 or LGP2-myc, followed by transfection with the titrated concentrations of poly(I:C) (D). Another 24 h later, the cells were harvested for luciferase assays. *p , 0.05, **p , 0.01, Student t test.

SVCV infection is easily inhibited by high doses of LGP2 additional activation of fish IFN promoters; however, in the late (Fig. 10A), because the inhibitory effect of LGP2, contrary to its phase of SVCV infection, the inhibition occurs when SVCV stimulatory potential, is proportional to its expression levels replicates in cells up to a relatively high titer that causes a far more (Fig. 6C). Moreover, time-course analyses showed that in the early significant induction of IFN expression than zebrafish LGP2 alone phase of virus infection, SVCV is a poor inducer due to its low (Fig. 10B). These results mean that the initial role of zebrafish replication levels and, concomitantly, zebrafish LGP2 functions as LGP2 in vivo should be positive at the beginning of SVCV in- a positive regulator because together with SVCV, it induces an fection, and its negative effect may only occur in the late phase. The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 10. Function switch of LGP2 from positive to negative regulation during virus infection. (A and B) EPC cells seeded in 24-well plates were transfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc (200 ng), together with different doses of LGP2. Then 24 h later, the transfected cells were infected 3 5 with SVCV of different titers for 24 h (A), or with SVCV of 10 TCID50 per ml or 10 TCID50 per ml for different times (B), and finally collected for luciferase assays. (C) EPC cells seeded in 24-well plates were transfected as in (A), with LGP2, LGP2v1, or LGP2v2 (100 ng) for 24 h, followed by SVCV 6 infection or GCRV infection (5 3 10 TCID50 per ml) for 24 h, and finally collected for luciferase assays. (D) EPC cells seeded in 24-well plates were transfected as in (A), with RIG-I-DN and MDA5-DN individually or collectively (100 ng each) for 24 h, followed by SVCV infection or GCRV infection for 24 h, and finally collected for luciferase assays. **p , 0.01, Student t test.

Therefore, whether zebrafish LGP2 exerts a positive or a neg- of low concentrations of poly(I:C) (16), but inhibits promoter ative role depends on SVCV titers, exactly on the balance of activation in the presence of large concentrations of poly(I:C) (9), immunostimulatory potential between LGP2 and SVCV at this which is consistent with the function analysis of zebrafish LGP2 in moment. In mammals, several studies have suggested that the the current study (Fig. 9). Considering the conserved structure of cellular LGP2 expression level is a key factor for regulation of a vertebrate LGP2, its function is likely conserved in fish and switch between positive and negative roles in RLR signal trans- mammals. If this is true in mice, it is easy to understand why duction (15, 16, 18, 19). This conclusion is derived from function LGP2-deficient mice are more susceptible to virus infection (12, analysis of LGP2 on MDA5-mediated IFN signaling in the pres- 13), because they have lost the initially positive regulation of ence of poly(I:C), particularly at limited levels (16, 18, 19). No- LGP2 in the early phase of virus infection. Our results might tably, these studies have also shown that human LGP2 provide a basis for further study of mammalian LGP2 function significantly promotes IFN-b promoter activation in the presence in vitro. 14 DIFFERENTIAL FUNCTION OF ZEBRAFISH LGP2 SPLICING TRANSCRIPTS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 11. Differential regulation of IFN response by three zebrafish LGP2 splicing forms individually and collectively. EPC cells seeded in 24-well plates were cotransfected with DrIFN-w1pro-luc or DrIFN-w3pro-luc (200 ng), control Renilla luciferase plasmid pRL-TK (20 ng), in the presence of LGP2, LGP2v1, or LGP2v2 plasmids at increasing amounts (A–C) or at a fixed dose of 200 ng (D). Then 24 h later, the cells were transfected again without (A and D) or with poly(I:C) at 4 ng/ml (B)or1mg/ml (C) for another 24 h, and then harvested for detection of luciferase activity. The data shown are representative of two independent experiments, each performed in triplicate.

Despite the bilateral function of zebrafish LGP2 on SVCV in- can activate IRF3 and IFN transcription (43). Interestingly, solid fection, we cannot exclude other possible mechanisms of fish LGP2 evidence has shown that grouper LGP2 and grass carp LGP2 against different viruses. For example, human LGP2 selectively negatively regulate IFN response against virus infection in vitro downregulates IFN response by seasonal influenza A viruses that (32, 33). However, it should be pointed out that in the current The Journal of Immunology 15 study overexpression of zebrafish LGP2-myc, a tag-fused LGP2 IFN expression (Fig. 6D). A puzzling question is how LGP2 utilizes plasmid, cannot potentiate IFN promoter activation, but its inhibi- a set of RLR signaling molecules to exert a disparate function in the tory function retains intact (Fig. 9D). The same happens to the different phases of virus infection. Similar to the above-described MITA gene, as evidenced by the findings that tag-fused fish and mechanism for function switch of zebrafish LGP2 on transfected mouse MITA proteins lose their antiviral activity but display a poly(I:C)- or SVCV-triggered IFN signaling, it is likely that the dominant negative effect (27, 44). In addition, although GCRV, a balance of stimulatory potential between zebrafish LGP2 and a dsRNA virus (45), induces IFN response through RIG-I and MDA5 given signaling factor in a specific cell state ingeniously determines (Fig. 10D), we failed to detect an obvious inhibitory effect of which role zebrafish LGP2 has to play. zebrafish LGP2 on IFN promoter activation by GCRV (Fig. 10C). In summary, the data in the current study seemingly resolve the This is probably not the case, because in some separate assays we puzzles in fish LGP2. It is likely that zebrafish LGP2 is expressed at have indeed observed the inhibitory effect of zebrafish LGP2 (data low levels in the beginning of virus infection, thus functioning as a not shown). A reasonable explanation is that it is easy to titer poly(I:C) positive regulator of IFN signaling, but in the late phase of virus to handle its immunostimulatory levels, but due to the complex infection it switches to a negative role. At low expression levels, interaction between fish cells and a given virus, it is hard to test an zebrafish LGP2 has more stimulatory potential than MDA5 and appropriate point of virus titer or infection duration, at which RIG-I (Fig. 5D, 5E); therefore, LGP2 is likely a master activator zebrafish LGP2 always plays a negative role. The same is seen for of IFN innate antiviral response in the early time of virus infec- LGP2v1 and LGP2v2 on activation both fish IFN promoters under tion, because at this time, zebrafish RIG-I and MDA5 are also SVCV infection (Fig. 10C) and for LGP2 on DrIFN-w3 promoter expressed at low levels (Fig. 2E) and have a weaker stimulatory activation by SVCV (Fig. 10A). Considering the differential sen- potential than LGP2 (Fig. 5E). Nevertheless, the maximum Downloaded from sitivity of DrIFN-w1 and DrIFN-w3 promoters to LGP2 and a rel- stimulatory effect of LGP2 is less than that of MDA5 and RIG-I atively poor inhibitory effect of LGP2v1 and LGP2v2 (Figs. 9, 10), along with the increase of their expression levels, and in addition this inhibition would occur if the expression levels of LGP2, to the role of LGP2 in IFN response, LGP2 has the potential to LGP2v1, and LGP2v2 were very high, although it is hard to operate control CD8+ T cell survival and fitness against divergent RNA due to limited capacity of transfection. If this is true, the in vivo viruses (13). Therefore, the antiviral effect of LGP2 observed in

inhibitory effects of LGP2v1 and LGP2v2 may not be relevant due knockout mice should be attributable to its multiple immunoactive http://www.jimmunol.org/ to their weak expression levels under virus infection. properties in innate immunity and adaptive immunity. The unique structure of LGP2 proteins, which lack N-terminal CARD domains, makes it difficult to understand how they trigger Disclosures IFN antiviral response as RIG-I and MDA5 do. In mammals, whereas The authors have no financial conflicts of interest. initial experiments do not reveal an ability of LGP2 to activate IFNb promoter and inhibit virus replication (7, 8), subsequent studies have obtained the opposite results (12, 46). In these cases, LGP2- References € directed ISRE stimulation in 293T cells might be attributable to its 1. Gurtler, C., and A. G. Bowie. 2013. Innate immune detection of microbial nucleic acids. Trends Microbiol. 21: 413–420. association with endogenous RLRs that contain CARDs (46). Direct 2. 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