Unique Composition of Intronless and Intron-Containing Type I Ifns in The

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent This information is current as Retroposition Hypothesis for Type I IFN of September 28, 2021. Genes in Amphibians Zhen Gan, Yue Cong Yang, Shan Nan Chen, Jing Hou, Zubair Ahmed Laghari, Bei Huang, Nan Li and Pin Nie Downloaded from J Immunol published online 2 November 2018 http://www.jimmunol.org/content/early/2018/11/01/jimmun ol.1800553 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/11/01/jimmunol.180055 Material 3.DCSupplemental

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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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 2, 2018, doi:10.4049/jimmunol.1800553 The Journal of Immunology

Unique Composition of Intronless and Intron-Containing Type I IFNs in the Tibetan Frog Nanorana parkeri Provides New Evidence To Support Independent Retroposition Hypothesis for Type I IFN Genes in Amphibians

Zhen Gan,*,† Yue Cong Yang,*,† Shan Nan Chen,* Jing Hou,*,† Zubair Ahmed Laghari,* Bei Huang,* Nan Li,* and Pin Nie*,‡,x

In vertebrates, intron-containing and intronless type I IFN genes have recently been reported in amphibian model species Xenopus tropicalis and X. laevis. However, whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes

or just represent the independent divergence in amphibians is unknown or even uninvestigated. In this study, both intron- Downloaded from containing and intronless type I IFN genes, as well as their receptor genes, were identiﬁed in the Tibetan frog Nanorana parkeri. The evidence obtained from homology, synteny, phylogeny, and divergence time showed that intronless type I IFN genes in N. parkeri and in Xenopus might have arisen from two independent retroposition events occurred in these two lineages, and the retrotransposition causing the generation of intronless type I IFN genes in amniotes is another independent event beyond the two in amphibians. It can then be proposed that intronless type I IFNs in N. parkeri and Xenopus may not be the ancestral genes of

intronless type I IFNs in amniotes but may just represent two independent bifurcations in the amphibian lineage. Furthermore, http://www.jimmunol.org/ both intronless and intron-containing type I IFNs in N. parkeri showed strong ability in inducing the expression of IFN-stimulated genes and the strong antiviral activity against frog virus 3. The present study thus provides the evolutionary evidence to support the independent retroposition hypothesis for the occurrence of intronless type I IFN genes in amphibians and contributes to a functional understanding of type I IFNs in this group of vertebrates. The Journal of Immunology, 2018, 201: 000–000.

nterferons are a family of pleiotropic cytokines with antivi- From an evolutionary point of view, type I IFNs have been ral, antitumor, and immunoregulatory functions in verte- identified in the classes of vertebrates from fish (teleost and car- brates (1–3). According to sequence similarity, receptor tilaginous fishes) to mammals (5, 6, 12–16). However, the detailed

I by guest on September 28, 2021 usage, and biological activity, IFNs are currently classified into phylogeny and evolutionary history of type I IFNs have not been three types [i.e., type I, type II, and type III IFNs (1–3)], among fully elucidated in vertebrates. An obvious difference among type which type I IFNs are considered as quintessential antiviral cy- I IFNs in vertebrates is their gene organization: type I IFNs in fish tokines because of their central roles in antiviral immune response are encoded by intron-containing genes with the genomic orga- (1, 4). In mammals, type I IFNs have at least seven major ho- nization of five exons and four introns (12, 13), whereas type I IFN mologous subgroups, including multigene a subtype with 13 genes in mammals, birds, and reptiles are intronless and are members in human and a single gene of each b, ε, k, t, d, and v expressed as single-exon transcripts (5, 6, 14). How and when the subtypes (5, 6). A striking feature of type I IFN signaling in primitive intron-containing type I IFN genes might have lost their mammals is the high redundancy of ligands, and all type I IFN introns and evolved into intronless type I IFN genes in amniotes members initiate signaling via the interaction with the same het- can be an intriguing question in the field of evolutionary immu- erodimeric receptor complex composed of IFNAR1 and IFNAR2 nology (12). (7, 8), thus leading to the transcription of several hundred Retroposition is recognized as a RNA-based duplication for the IFN-stimulated genes (ISGs) (9–11). creation of intronless duplicate genes in new genomic positions

*State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Address correspondence and reprint requests to Prof. Pin Nie, Institute of Hydro- Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province 430072, biology, Chinese Academy of Sciences, 7 South Donghu Road, Wuchang District, China; †University of Chinese Academy of Sciences, Beijing 100049, China; Wuhan, Hubei Province 430072, China. E-mail address: [email protected] ‡Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory The online version of this article contains supplemental material. for Marine Science and Technology, Qingdao, Shandong Province 266237, China; and xCollege of Marine Science and Engineering, Qingdao Agricultural University, Abbreviations used in this article: BI, Bayesian inference; CPE, cytopathic effect; Qingdao, Shandong Province 266109, China CRFB5, cytokine receptor family B–5; EPC, epithelioma papulosum cyprini; FNIII, ﬁbronectin type III; FOCAD, focadhesin; FV3, frog virus 3; HA, hemagglutinin; ORCIDs: 0000-0003-0009-3051 (Y.C.Y.); 0000-0002-9492-2940 (P.N.). HACD4, 3-hydroxyacyl-CoA dehydratase 4; HEK293T, human embryonic kidney Received for publication April 18, 2018. Accepted for publication September 17, 293T; hpi, hour postinjection; IFIT-5, IFN-induced protein with tetratricopeptide 2018. repeats 5; ISG, IFN-stimulated gene; ML, maximum likelihood; MYA, million years ago; NCBI, National Center for Biotechnology Information; NJ, neighbor-joining; This work was supported by a grant (31230075) from the National Natural Science Np-IFNAR1, predicted IFNAR1 in N. parkeri; ORF, open reading frame; pcDNA3.1, Foundation of China, by the China Agriculture Research System (CARS-46), and by pcDNA3.1/myc-His (2) A vector; pcDNA3.1–Np-IFN1, Np-IFN1 subcloned into special top talent plan One Thing One Decision (Yishi Yiyi) in Shandong Province, pcDNA3.1; pcDNA3.1–Np-IFNAR1, Np-IFNAR1 subcloned into pcDNA3.1; poly(I:C), China. polyinosinic/polycytidylic acid; rNp-IFN, recombinant Np-IFN. The sequences presented in this article have been submitted to the National Center for Biotechnology Information’s GenBank database (https://ncbi.nlm.nih.gov/genbank/) un- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 der accession numbers MG999514, MF346696, MG999515, MG999516, and MG999517.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800553 2 TIBETAN FROG TYPE I IFNs through the reverse transcription of parental genes (17). In a Fisher Scientific) to extract total RNA with TRIzol (Invitrogen). To analyze previous study to explore the possible molecular mechanism in the the expression of genes in N. parkeri after stimulation, 12 healthy individuals m generation of intronless type I IFNs, a hypothesis was proposed were each injected i.p. with polyinosinic/polycytidylic acid [poly(I:C);10 g/g body weight; Sigma-Aldrich] dissolved in amphibian PBS (pH 7.2) (21) and that intronless type I IFN genes in amniotes might have arisen were divided into four groups, each with three individuals. Twelve healthy from primitive intron-containing type I IFN genes via a retro- individuals as control were injected i.p. with an equal volume of amphibian position event when vertebrates migrated from aquatic to terres- PBS and were also equally divided into four groups. At 3, 6, 12, and 24 h trial environments (18). Now, this hypothesis has been generally postinjection (hpi), three animals in each group were sacrificed for the collection of organs/tissues, which were then placed in RNAlater (Thermo accepted (19, 20), but when this retroposition event might have Fisher Scientific) for extracting RNA with TRIzol. In addition, the muscle happened still remains unknown or uninvestigated. of healthy N. parkeri was taken and stored in ethanol for isolating the Interestingly, it is recently revealed that intronless and intron- genomic DNA. All animal protocols were performed following the Guide containing type I IFN genes coexist in two amphibian model spe- for the Care and Use of Laboratory Animals of the Chinese Academy of cies, the tropical clawed frog Xenopus tropicalis and the African Sciences and were approved by the Institute of Hydrobiology. The A6 cells (CCL-102) from American Type Culture Collection were clawed frog X. laevis (21, 22), implying that retroposition might cultured in 75% NCTC-109 medium (Life Technologies) supplemented with have occurred in amphibians, resulting in the presence of intronless 10% FBS (Life Technologies), 15% distilled water, 100 U/ml penicillin (Life type I IFN genes in amphibians, which have the same genomic or- Technologies), and 100 mg/ml streptomycin (Life Technologies) at 26˚C with ganization as the type I IFNs in amniotes. However, the phylogenetic 5% CO2. Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM (Life Technologies) with 10% FBS, 100 U/ml penicillin, and relationship between intronless type I IFNs in amphibians and am- 100 mg/ml streptomycin at 37˚C with 5% CO2. Epithelioma papulosum niotes has been heavily debated among comparative immunologists cyprini (EPC; GDC174) cells from China Center for Type Culture Collection

(20–22). Two different hypotheses, evolutionary continuum hy- were cultured in medium 199 (Life Technologies) supplemented with 10% Downloaded from pothesis and independent retroposition hypothesis, have been ten- FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 26˚C with 5% tatively proposed to explain the evolutionary position of type I IFNs CO2. FV3 (VR-567) from American Type Culture Collection was propa- gated in EPC cells as previously reported (26). in amphibians (20–22). Regarding the evolutionary continuum hypothesis, the retroposition event occurred in amphibians is regarded Cloning of type I IFN and their receptor genes in N. parkeri as the origin of all intronless type I IFN genes in vertebrates, and For the identification of type I IFN and their receptor genes in N. parkeri, all intronless type I IFNs in amphibians should be the ancestral genes of annotated type I IFN, IFNAR1, and IFNAR2 genes from the GenBank http://www.jimmunol.org/ intronless type I IFNs in amniotes (21, 22). But the evidence from database were used to construct a query set, and Basic Local Alignment phylogeny and synteny analyses of type I IFN genes in vertebrates Search Tool programs were used to search for all type I IFN, IFNAR1, and indicated that intronless type I IFN genes in Xenopus may not be the IFNAR2 genes in N. parkeri genome available in the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/ direct orthologs of type I IFN genes in amniotes, but may just rep- genome/36384). Putative transcripts were predicted from the genome se- resent an independent bifurcation of the type I IFNs in the Xenopus quences using the Fgenesh program (http://www.softberry.com). lineage (20, 21). To examine which of these two competing hy- The putative open reading frames (ORFs) of Np-IFN1, Np-IFNi1 and potheses is more applicable and rational, it is imperative to find new Np-IFNi2, and Np-IFNAR1 and Np-IFNAR2 were predicted by in silico evidence from other amphibian species (20–22). analysis. Using TRIzol Reagent, total RNA from spleen, kidney, and liver of N. parkeri treated for 12 h with poly(I:C) was extracted and mixed. The by guest on September 28, 2021 The Tibetan frog Nanorana parkeri is endemic to Qinghai– first-strand cDNA was synthesized from the above mixed total RNA using Tibetan Plateau and is the only another amphibian species whose RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific) and genome has been well sequenced and annotated except for Xenopus served as template for amplifying above mentioned genes. (23). In the current study, both intron-containing type I IFN, named To amplify genomic DNA sequences of Np-IFN1, Np-IFNi1, and Np-IFNi2, genomic DNA isolated from muscle of healthy N. parkeri using proteinase K as Np-IFN1, and intronless ones, Np-IFNi1 and Np-IFNi2, as well and phenol–chloroform extraction was served as template to amplify the as their receptor genes, Np-IFNAR1 and Np-IFNAR2, were iden- genomic DNA sequences of above listed genes by PCR with specific primers. tified in N. parkeri. The evidence obtained from homology, synteny, All PCR products were ligated into the pMD18-T vector (Takara Bio) and and phylogeny analyses, as well as from the estimation of diver- transformed into competent Escherichia coli cells. The positive clones were gence time, showed that intronless type I IFN genes in N. parkeri sequenced by a commercial company in Shanghai, China. The sequences of all PCR primers used in this study are summarized in Table I. and Xenopus might have arisen from two independent retroposition events that occurred in these two lineages, and the retrotransposition Sequence analyses of type I IFN and their receptor genes causing the generation of intronless type I IFN genes in amniotes is in N. parkeri another independent event beyond the two in amphibians. In con- The protein sequences were deduced from the nucleic acid sequences using the trast, the ability of these three Np-IFNs in inducing the expression program on the ExPASy Web site (http://ca.expasy.org/tools). Signal peptides of ISGs and in antiviral activity against the frog virus 3 (FV3) was were identified with the SignalP 4.1 server (www.cbs.dtu.dk/services/SignalP). also characterized in this study. To our knowledge, the current study N-glycosylation sites were analyzed by using pSite tool (http://linux1.softberry. represents the first discovery on the antiviral activity of intronless com/berry.phtml?topic=psite&group=programs&subgroup=proloc), and domains were predicted using the InterProScan program (http://www.ebi.ac. type I IFNs in amphibians against ranaviruses, the major viral uk/Tools/pfa/iprscan/). The secondary structure prediction was performed pathogens causing the worldwide amphibian population decline (24, with Scratch Protein Predictor (http://scratch.proteomics.ics.uci.edu/). 25). The present research thus provides the key evidence to support Homology between sequences was calculated using the MegAlign pro- the independent retroposition hypothesis for the occurrence of gram within the DNASTAR package. Chromosomal locations of type I IFN genes in N. parkeri, X. tropicalis, X. laevis, green anole lizard (Anolis intronless type I IFN genes in amphibians and contributes to a better carolinensis), painted turtle (Chrysemys picta bellii), Chinese alligator understanding of amphibian antiviral immunity. (Alligator sinensis), chicken (Gallus gallus), African ostrich (Struthio camelus), emperor penguin (Aptenodytes forsteri), human (Homo sapiens), mouse (Mus musculus), black flying fox (Pteropus alecto), and gray short- Materials and Methods tailed opossum (Monodelphis domestica) were obtained from the Sequence Animals, cells, and virus Viewer (https://www.ncbi.nlm.nih.gov/projects/sviewer/). All sequences used in the analysis are listed in Supplemental Table I. Adults of the Tibetan frog N. parkeri were collected from a natural water body in the Qinghai–Tibetan Plateau with the permission from a local au- Phylogenetic analysis of type I IFN genes in N. parkeri thority and the Institute of Hydrobiology, Chinese Academy of Sciences. To analyze the expression of target genes in healthy N. parkeri, organs/tissues Automated multiple sequence alignments for amino acids were generated were collected from three animals and were placed in RNAlater (Thermo using the ClustalX program (27), and nonconserved protein sequences of The Journal of Immunology 3 extralong chains at N- or C-terminals were removed manually. Structure- (Invitrogen). Forty-eight hours after the transfection, the supernatants of guided alignment (28, 29) was performed manually with the information of transfected HEK293T cells were collected and concentrated with a available three-dimensional structures of the type I IFNs, including human Microcon-10kDa molecular mass cutoff filter unit (MilliporeSigma). The IFN-a2andIFN-b (30, 31) and zebrafish IFN1 and IFN2 (28), and with the presence of recombinant Np-IFN (rNp-IFN)1, Np-IFNi1 (rNp-IFNi1), and information of predicted secondary structures of other type I IFNs generated Np-IFNi2 (rNp-IFNi2) in the supernatants of transfected HEK293T cells by Scratch Protein Predictor (http://scratch.proteomics.ics.uci.edu/). ProtTest was confirmed by Western blotting with a mouse anti–HA-tag mAb software (32) was used to select the best-fit model, which was the JTT model (Sigma-Aldrich), and densitometric calculation of immunoblotting bands for the construction of the phylogenetic tree of type I IFNs in vertebrates. was conducted using Image Lab Software (version 4.1). The rNp-IFN1, Bayesian inference (BI) analysis was carried out using MrBayes software rNp-IFNi1, and rNp-IFNi2 in the supernatants of transfected HEK293T (33), which was run for 100,000 generations with 100-sample frequency. Set cells were diluted to the same concentration by using DMEM. After the value of burn-in was 2000, and IL-10 from human was used as outgroup. addition of a protease inhibitor mixture (Roche), the supernatants of Maximum likelihood (ML) and neighbor-joining (NJ) analyses were per- transfected HEK293T cells were aliquoted and stored at 280˚C until formed to construct trees with 1000 bootstrap replicates using PhyML further use. software (34) and MEGA7.0 (35), respectively. All sequences used in the analysis are listed in Supplemental Table I. Examination of ISG expression induced by type I IFNs from N. parkeri Estimation of divergence times of intron-containing and intronless type I IFN genes in N. parkeri and Xenopus At present, any cell line derived from N. parkeri is not available. The A6 cells, a kind of kidney fibroblast cell line from X. laevis, which is routinely The divergence time of intron-containing and intronless type I IFN genes in used for studying the antiviral immunity in amphibians (26, 45, 46), was N. parkeri and Xenopus was estimated using the RelTime method, which used to analyze the expression of ISGs induced by type I IFNs from has been shown to perform well in the calculation of divergence times for N. parkeri in the current study. Briefly, 1 3 106 A6 cells were seeded into

duplicated genes (36, 37). Briefly, amino acid sequences of type I IFNs individual wells of 24-well plates and transfected with empty pcDNA3.1 Downloaded from from N. parkeri, X. tropicalis, X. laevis, D. rerio, A. carolinensi, G. gallus, vector as control, or with pcDNA3.1–Np-IFN1, –Np-IFNi1 and –Np- H. sapiens, and basking shark (Cetorhinus maximus) were aligned by IFNi2, respectively, following the protocol of FuGENE HD Transfection structure-guided alignment method as described above, and nucleotide Reagent (Promega). Forty-eight hours after transfection, the cells were sequences of these type I IFN genes were aligned according to the result of harvested with RNA extracted to detect the expression of ISGs, including structure-guided amino acid sequence alignment. Then, all positions MX1, Viperin, and IFN-induced protein with tetratricopeptide repeats 5 containing gaps and missing data in nucleotide sequence alignment were (IFIT-5) (47, 48). Specific primers (Table I) were designed for target genes eliminated, and the ML tree was constructed with the Tamura–Nei model in X. laevis based on the sequences from NCBI. The experiments were of nucleotide substitutions (38). The TimeTree was computed using three performed for three biological replicates with three technical replicates for http://www.jimmunol.org/ calibration constraints. One calibration was based on the assumption that each biological replicate. The expression of target genes was normalized teleosts and tetrapods were diverged at ∼425–446 million years ago against the level of b-actin, and fold changes were calculated relative to (MYA) (39), secondary calibration date was referred to reptiles and control group. mammals diverged at ∼297–326 MYA (39, 40), and third calibration date To verify whether Np-IFNs were associated with Np-IFNAR1 and Np- referred to N. parkeri and Xenopus diverged at ∼187–220 MYA (39, 41). A IFNAR2, 1 3 106 A6 cells were seeded into wells of 24-well plates and type I IFN gene from basking shark was used as outgroup. Finally, di- cotransfected separately with expressing plasmids of each of the three vergence times of inferred clades were visualized by MEGA7.0 (35). All Np-IFNs in combination with Np-IFNAR1 subcloned into pcDNA3.1 sequences used in the analysis are listed in Supplemental Table I. (pcDNA3.1–Np-IFNAR1) or pcDNA3.1–Np-IFNAR2 or the two together. Forty-eight hours after transfection, the cells were harvested, and RNA was Quantitative analysis of type I IFN mRNA expression extracted to detect the expression of ISGs as described above. by guest on September 28, 2021 in N. parkeri To analyze the expression of ISGs induced by recombinant type I IFNs from N. parkeri,13 106 A6 cells were seeded into each well of 24-well Total RNA from organs/tissues of different individuals was used separately plates and cotransfected separately with different combinations of for the synthesis of the first-strand cDNA sequence using RevertAid First- pcDNA3.1–Np-IFNAR1 and –Np-IFNAR2. Forty-eight hours after trans- Strand cDNA Synthesis Kit (Thermo Fisher Scientific) by following the fection, the cells were incubated for 10 h with 200 ml supernatant of manufacturer’s instruction. Specific primers (Table I) were used to amplify transfected HEK293T cells containing rNp-IFN1, rNp-IFNi1, or rNp- Np-IFN1, Np-IFNi1, Np-IFNi2, and b-actin (MF346697) fragments by IFNi2 and an equal volume of the vector control, respectively. The cells PCR. All PCR products were sequenced as described above. Sequenced were harvested, and RNA was extracted to detect the expression of ISGs as plasmid DNA of positive clones was extracted with Plasmid Mini Kit described above. (Omega Bio-tek) and measured using spectrophotometer (NanoDrop 8000; Thermo Fisher Scientific). The copy number was calculated following a Assays for antiviral activity of type I IFNs from N. parkeri method described previously (21). To establish a standard curve, plasmids against FV3 were diluted in serial 10-fold dilutions ranging from 1026 to 1022 before being quantified using PCR with CFX96 Real-Time PCR Detection Sys- To analyze the antiviral capacity of type I IFNs from N. parkeri against tem (Bio-Rad). A final volume of 20 ml PCR reaction system contained FV3, a kind of ranavirus that is used for studying host/virus interaction in 10 ml iQ SYBR Green Supermix (Bio-Rad), 1 ml of each primers, 7 mlof amphibians (45, 49), 1 3 106 A6 cells were seeded into each well of 24- sterile water, and 1-ml cDNA template with the PCR protocol as the fol- well plates and then cotransfected separately with empty pcDNA3.1 vector lowing: one cycle of 95˚C for 3 min, followed by 45 cycles of 95˚C for (as control), expressing plasmids of each of the three Np-IFNs, or together 10 s, 56–58˚C for 20 s, and 72˚C for 40 s. Each sample was run with three with single or two receptor-expressing plasmids, pcDNA3.1–Np-IFNAR1 technical replicates, and the gene expression for each sample was nor- and Np–IFNAR2, following the protocol of FuGENE HD Transfection malized against b-actin. Data analysis was performed using the 22DDCt Reagent. Forty-eight hours after transfection, the cells were infected for method (42), and fold changes were calculated relative to control group. 72 h with FV3 at the multiplicity of infection of 0.3. The culture supernatants were removed, and A6 cell monolayers were fixed in 10% para- Production of recombinant type I IFNs from N. parkeri formaldehyde for 1 h before being stained with 0.05% crystal violet for the observation of cytopathic effect (CPE), as previously reported (50). For the production of recombinant type I IFNs in N. parkeri, Np-IFN1–, To assess the effect of type I IFNs from N. parkeri on FV3 burdens, A6 Np-IFNi1–, and Np-IFNi2–expression plasmids were transfected sepa- cells were cotransfected with a variety of plasmids, as described above, rately into HEK293T cells as previously reported, with minor modification followed by FV3 infection at an multiplicity of infection of 0.3 for 72 h. (15, 22, 43, 44). Briefly, the entire ORF of Np-IFN1, Np-IFNi1, and Np- Then, A6 cells were subjected to three rounds of sequential freeze/thaw lysis, IFNi2 was amplified and inserted with the coding sequence of hemag- with the resulting homogenates collected for the determination of virus titer glutinin (HA)-tag between the last protein-coding codon, and stop one for by standard plaque assays, which was performed on EPC cell monolayers Np-IFN1, Np-IFNi1, and Np-IFNi2 genes, before being subcloned into under an overlay of 0.5% methylcellulose, as previously reported (50). pcDNA3.1/myc-His (2) A vector (pcDNA3.1; Invitrogen): Np-IFN1 subcloned into pcDNA3.1 (pcDNA3.1–Np-IFN1), Np-IFNi1 subcloned Statistical analysis into pcDNA3.1 (pcDNA3.1–Np-IFNi1), and pcDNA3.1–Np-IFNi2. Then pcDNA3.1–Np-IFN1, –Np-IFNi1, –Np-IFNi2, and empty pcDNA3.1 Statistical analyses were conducted on three repeated experiments, with vector (as control) were transfected into HEK293T cells, respectively, one-way ANOVA carried out in SPSS 17.0. Data were presented as mean 6 following the protocol of Lipofectamine 2000 Transfection Reagent SE, and statistical significance was defined as p , 0.05. 4 TIBETAN FROG TYPE I IFNs

Results Interestingly, the Xt-IFNi1 (Xt-IFNi1.2, Xt-IFNi2.1, and Xt- Sequence analysis of type I IFN genes in N. parkeri IFNi3.1) share very low sequence identity (23.3–26.6%) with their intron-containing progenitor, Xt-IFN5, whereas Np-IFNi1 By searching the N. parkeri genome, Np-IFN1 was identified on and Np-IFNi2 share relatively high sequence homology (64.6– Scaffold 348 (NCBI Reference Sequence: NW_017306616.1), and 65.6%) with Np-IFN1 (Table II). Np-IFNi1 and Np-IFNi2 were identified on Scaffold 1059 To test if the predicted genomic structure of Np-IFNs through (NW_017307327.1) and Scaffold 2618 (NW_017308886.1), re- in silico analysis is accurate, the genomic DNA sequences of spectively. To test whether predicted type I IFN genes Np-IFN1, Np-IFNi1, and Np-IFNi2 were cloned from N. parkeri are expressed in vivo at transcription level, the ORF sequences of for comparing the cDNA and genomic DNA sequences of these Np-IFN1, Np-IFNi1, and Np-IFNi2 were cloned from N. parkeri genes. It is confirmed that Np-IFNi1 and Np-IFNi2 are intronless with the following accession numbers in NCBI’s GenBank (https:// genes, whereas Np-IFN1 is an intron-containing IFN with five- www.ncbi.nlm.nih.gov/genbank/): MG999514, MF346696, and exon and four-intron genomic organization. MG999515, respectively. In addition, two intronless type I IFN fragments, Np-IFNi-f1 and Np-IFNi-f2, linked to Np-IFNi1 were Sequence analysis of type I IFN receptor genes in N. parkeri found on Scaffold 1059, but these two fragments could not be By searching the N. parkeri genome, Np-IFNAR1 and Np-IFNAR2 detected by either conventional or rapid amplification of cDNA were identified on Scaffold 817 (NW_017307085.1) and Scaffold ends PCR on cDNA templates derived from healthy, as well as 805 (NW_017307073.1), respectively. To test whether predicted immunologically stimulated, frogs. type I IFN receptor genes are expressed in vivo at the level of

The ORFs of Np-IFN1, Np-IFNi1, and Np-IFNi2 are 570, 561, transcription, the ORF sequences of Np-IFNAR1 and Np-IFNAR2 Downloaded from and 576 bp in size, which encode a protein of 189, 186, and 191 aa, were cloned from N. parkeri with the following accession numbers respectively. Although Np-IFN1, Np-IFNi1, and Np-IFNi2 share in NCBI’s GenBank (https://www.ncbi.nlm.nih.gov/genbank/): relatively low identity, being 15.2–41.3, 15.3–20.9, 16.3–16.9, MG999516 and MG999517, which are 999 and 1482 bp in size, 18.5–21.9, and 11.8–19.4% with type I IFNs in X. tropicalis, encoding a protein of 332 and 493 aa, respectively. As the members human, chicken, green anole lizard, and zebraﬁsh, respectively of the class II cytokine receptors, the predicted proteins of Np- (Table II), two important structural characteristics known in type I

IFNAR1 and Np-IFNAR2 both contain an extracellular region http://www.jimmunol.org/ IFNs are conserved in predicted proteins of above Np-IFNs, in- with a putative signal peptide, a transmembrane region and an including a predicted signal peptide and four cysteine residues tracellular region (Supplemental Fig. 1). participated in the formation of disulfide bonds (51) (Fig. 1). N- As expected, two important structural features known in glycosylation sites, which are involved in posttranslational mod- IFNAR1 are conserved in the predicted protein of Np-IFNAR1, ifications of type I IFNs (52), are conserved in predicted proteins including four cysteine residues involved in the formation of of Np-IFN1 and Np-IFNi2, but are not present in the predicted two disulfide bonds and the DSGXY motif, which is vital for protein of Np-IFNi1 (Fig. 1). Using Predict Secondary Structure ubiquitination, endocytosis, and degradation of IFNAR1 (7, 8, 53) program, it is revealed that five a-helices are present in predicted (Supplemental Fig. 1A). Notably, unlike the extracellular region of proteins of Np-IFN1, Np-IFNi1, and Np-IFNi2, and their sequence IFNAR1 in mammals and birds that contains four fibronectin type by guest on September 28, 2021 features and locations are respectively similar to helices A, B, C, III (FNIII) domains (7, 8), the extracellular region of Np-IFNAR1 D, and E of IFN-a and IFN-b in human (30, 31) (Fig. 1). contains only two FNIII domains, which is similar to cytokine

Table I. PCR primers used in this study

Primer Sequence 59→39 Purpose Np-IFN1–O-F/R 59-ATGGCCGCCACGTATTCCTGGA/TTAGAAAAGATGTCTCTGTAGTCGC-39 ORF cloning Np-IFNi1–O-F/R 59-ATGACCAGCACATCTTCCTGGAC/TTAGTCATGTGACTTCTGGTTTCTC-39 Np-IFNi2–O-F/R 59-ATGACCAGCACATCTTCCTGGAC/TTACTGAGCTGATTGCTGATCATGTG-39 Np-IFNAR1–O-F/R 59-ATGGTCAGGGGCCCAGGGGTCC/TCATTGAGTGGCGGCTGTGACGTC-39 Np-IFNAR2–O-F/R 59-ATGGCAAGCTTGATGCTTCTTTTA/TCATCTTCTCATGTACCCTGAAA-39 Np-b-actin–O-F/R 59-ATGGAAGATGATATCGCCGCCC/TTAAAAGCATTTGCGGTGGACAA-39 Np-IFN1–G-F/R 59-ATGGCCGCCACGTATTCCTGGA/TTAGAAAAGATGTCTCTGTAGTCGC-39 Genomic sequence cloning Np-IFNi1–G-F/R 59-ATGACCAGCACATCTTCCTGGAC/TTAGTCATGTGACTTCTGGTTTCTC-39 Np-IFNi2–G-F/R 59-ATGACCAGCACATCTTCCTGGAC/TTACTGAGCTGATTGCTGATCATGTG-39 Np-IFN1–HA-F/R 59-CCGCTCGAGATGGCCGCCACGTATTCCTGGACAG/CCGGAATTCTTAAGC-39 Construction of eukaryotic 59-GTAATCTGGAACATCGTATGGGTAGAAAAGATGTCTCTGTAGT-39 expression vector Np-IFNi1–HA-F/R 59-ATGACCAGCACATCTTCCTGGAC/TTAAGCGTAATCTGGAACATCGTATGG-39 59-GTAGTCATGTGACTTCTGGTTTCTCATG-39 Np-IFNi2–HA-F/R 59-CCGCTCGAGATGACCAGCACATCTTCCTGGACGG/CCGGAATTCTTAAGCG-39 59-TAATCTGGAACATCGTATGGGTACTGAGCTGATTGCTGATCA-39 Np-IFNAR1–E-F/R 59-CGCGGATCCATGGTCAGGGGCCCAGGGGTCCTCC/CCCAAGCTTTCATTGA-39 59-GTGGCGGCTGTGACGTCA-39 Np-IFNAR2–E-F/R 59-CCGCTCGAGATGGCAAGCTTGATGCTTCTTTTAA/CCGGAATTCTCATCTTC-39 59-TCATGTACCCTGAAACA-39 Np-b-actin–Q-F/R 59-AGATGATGCTCCTCGTGCTGT/TCTTCTCTCGGTTTGCTTTGG-39 Real-time PCR Np-IFN1–Q-F/R 59-GGCATCTCTACCAAATCACCG/CCTCGTCTTCCTGCTCCTCTAG-39 Np-IFNi1–Q-F/R 59-CAACTCACCTCACCTTCCTGTC/CAGTGTCCTTCTGCTCCTCTAG-39 Np-IFNi2–Q-F/R 59-CAACTCACCTCACCTTCCTGTC/GCATTGTCTTTCTGCTCCTCTAG-39 A6-b-actin–Q-F/R 59-CCCCGTGCTGTTTTCCCATC/TGGTGCCACTCGCAGTTCAT-39 A6-MX1–Q-F/R 59-AGCATTTTTTGGCATTTCCCA/TTCGTTGAGTCGTCCGCTGTA-39 A6-Viperin–Q-F/R 59-ATGTTCTCTGCTGTTTGGGGGA/GTTTTACGCTCTGGGTTGGCG-39 A6-IFIT-5–Q-F/R 59-TACTTCTGGTTTTCTCCATCATCA/ATGACTTTTTCTCTTCTACCGCCT-39 The Journal of Immunology 5

receptor family B–5 (CRFB5), the homolog of IFNAR1 in ﬁsh (54–56) (Supplemental Fig. 1A). Similar to the counterparts in mammals and ﬁsh, Np-IFNAR2 19.5 has a longer intracellular region than Np-IFNAR1 (Supplemental Fig. 1B). In addition, several important structural features known in IFNAR2 are conserved in the predicted protein of Np- IFNAR2, including two FNIII domains in the extracellular re-

22.2 12.3 gion, four cysteine residues participated in the formation of two disulﬁde bonds, and the last tyrosine residue (Y510 in human), which plays a critical role in the activation of STAT1 and STAT2 (7, 8, 57) (Supplemental Fig. 1B).

20.8 20.5 22.0 Syntenic analysis of type I IFNs in vertebrates Ac-IFN1 Dr-IFN1 Dr-IFN2 On the chromosome 3 of X. tropicalis, intronless type I IFNs are adjacent to glutaryl-CoA dehydrogenase (GCDH); lectin,

a galactoside-binding, soluble, 9 (LGALS9); RAB3D, member RAS oncogene family (RAB3D); and transmembrane protein 23.5 23.0 16.7 20.2

Gg-IFN- 205 (TMEM205; Fig. 2A). X. laevis is an allotetraploid that has

two homologous subgenomes, with one chromosome (called Downloaded from chromosome L) maintaining the ancestral state and the another b (chromosome S) experiencing much more rearrangement (58); 8.9 17.4 14.0 14.9 9.8 17.0 15.2 15.2 20.6 11.8 32.8 20.3 22.3 21.7 23.2 11.4 10.7 11.3 15.0 11.9 11.0 13.5 10.1 16.0 12.6 17.615.3 16.9 16.3 20.2 21.9 19.4 19.4 13.0 11.8 15.3 16.9 18.5 18.8 13.6 so, the intronless type I IFNs on the chromosome 3L are adjacent to the same genes as in X. tropicalis, whereas intronless type I IFNs on the Scaffold 20 are linked to calreticulin homeolog

S (CALR.S), phenylalanyl-tRNA synthetase alpha subunit http://www.jimmunol.org/ 2 Hs-IFN-

a S homeolog (FARSA.S), RAB3D S homeolog (RAB3D.S), and

16.4 thyroid hormone–induced bZip protein S homeolog (THIBZ.S; Fig. 2A). By contrast, on the Scaffold 1059 of N. parkeri genome, Np- IFNi1 and two intronless type I IFN fragments Np-IFNi–f1 and Np-IFNi–f2 are not adjacent to the above genes, but tightly

22.3 10.2 linked to distinct genes, including receptor-type tyrosine/protein

Amino Acid Identity (%) phosphatase k, extensin-1, and complement receptor type 1

(CR1; Fig. 2A). Np-IFNi2 on the Scaffold 2618 is adjacent to by guest on September 28, 2021 LOC108802036 and erythroid membrane-associated protein-like (Fig. 2A), which may be attributed to the independent chromo- 24.1 25.1 12.7 24.4 26.6 14.1 15.516.1 40.3 41.3 20.9 18.6 15.2 38.6 19.2 somal rearrangement in N. parkeri or incomplete sequence

. assembly. In reptiles, such as in green anole lizard, the genes around intronless type I IFN gene locus are focadhesin (FOCAD), 3-

X. tropicalis hydroxyacyl-CoA dehydratase 4 (HACD4), methylthioadenosine 25.6 28.0 42.4 27.4 phosphorylase (MTAP), and cyclin dependent kinase inhibitor 2b ;Xt, (CDKN2B; Fig. 2B). Intronless type I IFNs in painted turtle and Chinese alligator are located at the same locus as in green anole lizard, because these genes are also linked to FOCAD and HACD4 (Fig. 2B). In birds, such as in chicken, African ostrich, N. parkeri ;Np, 22.0 26.0 22.0 and emperor penguin, only one type I IFN gene is adjacent to FOCAD and HACD4, and most intronless type I IFN genes are linked to nucleolar protein 6 (NOL6), ubiquitin-conjugating H. sapiens enzyme E2 R2 (UBE2R2), DDB1- and CUL4-associated factor

;Hs, 12 (DCAF12), and ubiquitin-associated protein 1 (UBAP1;

90.3 Fig. 2C). In mammals, such as in human, mouse, black ﬂying fox, and gray short-tailed opossum, intronless type I IFNs are adjacent to the same genes as in green anole lizard (Fig. 2D).

G. gallus ;Gg, These data revealed that intronless type I IFN loci may not be syntenic in Xenopus, N. parkeri, and amniotes. D. rerio

Np-IFNi1 Np-IFNi2 Xt-IFNi1.2 Xt-IFNi2.1 Xt-IFNi3.1 Xt-IFN5 Hs-IFN- Phylogenetic relationship of type I IFNs in vertebrates ;Dr, To understand the phylogenetic relationship among intronless and

2 intron-containing type I IFNs in N. parkeri and Xenopus, and type a I IFNs in ﬁsh and amniotes, BI, ML, and NJ trees were constructed

A. carolinensis using protein sequences (Fig. 3, Supplemental Fig. 2). In general, Xt-IFNi3.1 Xt-IFN5 Xt-IFNi2.1 Hs-IFN- a Np-IFNi1 Np-IFNi2 Xt-IFNi1.2 Np-IFN1 65.6 64.6 Hs-IFN- b Gg-IFN- Ac-IFN1 Dr-IFN1

Ac, the BI, ML, and NJ trees showed almost identical topology. Un-

Table II. Amino acid identity among type I IFNs in Tibetan frog, tropical clawed frog, human, chicken, green anole lizard, and zebraﬁsh expectedly, intronless type I IFNs in N. parkeri and Xenopus were 6 TIBETAN FROG TYPE I IFNs Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Multiple alignments of deduced type I IFN protein sequences in N. parkeri. Identical amino acids are indicated with an asterisk (*), and amino acids with high and low similarity are indicated with a colon (:) and a period (.), respectively. The putative signal peptides and cysteine residues that participated in the formation of disulfide bonds are underlined and bolded, respectively. The amino acid sequences from different species are named accordingly and abbreviated. The a-helices (A–E) for IFN-a and IFN-b in human are highlighted in solid lines above the alignment, and corresponding sequences are illustrated in light gray shadow. Deep gray shadow sequences indicate predicted a-helices of type I IFNs in N. parkeri. Dr, zebrafish Danio rerio; Gg, chicken G. gallus; Mm, mouse M. musculus; Hs, human H. sapiens; Np, Tibetan frog N. parkeri; Xt, tropical clawed frog X. tropicalis. not grouped together; but intronless and intron-containing type I phylogeny tree of type I IFN genes in these two lineages was IFNs in N. parkeri were grouped together with the support of high constructed by the RelTime method. As shown in Fig. 4, intronless bootstrap value, which was then clustered together with the intron- and intron-containing type I IFN genes in Xenopus might have di- containing type I IFN subgroup II in Xenopus. All intronless type I verged at ∼180.70 MYA, whereas the divergence of intronless type IFNs in Xenopus were grouped together to form a separate clade, I IFN genes and their intron-containing progenitors in N. parkeri which was in turn, clustered with the clade containing type I IFNs in might have occurred at ∼87.57 MYA. In addition, the emergence N. parkeri and intron-containing type I IFN subgroup II in Xenopus. time of intronless type I IFN genes in amniotes is ∼310.65 MYA. However, intron-containing type I IFN subgroup I in Xenopus and Interestingly, IFN-a and IFN-b might have diverged at ∼252.09 type I IFNs in fish and amniotes were clustered into a major clade at MYA in mammals and ∼36.76 MYA in birds, respectively. the base of the phylogenetic tree, which is probably the evolutionary Expression pattern of type I IFN genes in N. parkeri continuum of type I IFNs in vertebrates. Interestingly, intron-containing type I IFNs in Ambystoma mex- In healthy N. parkeri, the mRNA of Np-IFN1, Np-IFNi1, and icanum were also clustered with the clade containing type I IFNs in Np-IFNi2 was detected in all organs/tissues examined, with the N. parkeri and intron-containing type I IFN subgroup II and highest level of Np-IFN1 observed in kidney, and the highest intronless type I IFNs in Xenopus (Fig. 3, Supplemental Fig. 2). of Np-IFNi1 and Np-IFNi2 in intestine (Fig. 5A). Following poly(I:C) stimulation, Np-IFN1, Np-IFNi1, and Np-IFNi2 all Divergence times of intron-containing and intronless type I showed a time-dependent increase at the transcript level in spleen, IFN genes in N. parkeri and Xenopus kidney, and liver (Fig. 5B), with the highest level all observed at To estimate the divergence time of intron-containing and intronless 12 hpi, except for Np-IFNi1, which had highest transcript level at type I IFN genes in N. parkeri and Xenopus, the time-calibrated 6 hpi (Fig. 5B). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 2. Gene synteny analyses of intronless type I IFN loci in amphibians (A), reptiles (B), birds (C), and mammals (D). All genes are indicated with arrow symbols pointing to the transcription direction. Intronless type I IFNs are indicated in red. Gray pentagons represent intronless type I IFN gene fragments, and gray boxes represent pseudogenes in human. GCDH and GCDH.L, blue; LGALS9 and LGALS9.L, light blue; RAB3D, RAB3D.L, and RAB3D.S, dark purple; and TMEM205 and TMEM205.L, light purple. CALR.S, FARSA.S, THIBZ.S, RTPK, Extensin-1, CR1, EMAP, LOC108802036, and KLHL9 are dyed using white. FOCAD, HACD4, and MTAP are marked using purple, pink, and dark green, respectively. CDKN2A and CDKN2B appear as light green. NOL6, UBE2R2, DCAF12, and UBAP1 are illustrated in deep yellow, orange, peach, and ruby red, respectively. CALR.S, calreticulin by guest on September 28, 2021 S homeolog; CDKN2A, cyclin dependent kinase inhibitor 2a; CDKN2B, cyclin dependent kinase inhibitor 2b; CR1, complement receptor type 1; DCAF12, DDB1- and CUL4-associated factor 12; EMAP, erythroid membrane-associated protein-like; FARSA.S, phenylalanyl-tRNA synthetase a subunit S homeolog; GCDH, glutaryl-CoA dehydrogenase; GCDH.L, GCDH L homeolog; KLHL9, kelch-like family member 9; LGALS9, lectin, galactoside- binding, soluble, 9; LGALS9.L, LGALS9 L homeolog; MTAP, methylthioadenosine phosphorylase; NOL6, nucleolar protein 6; RAB3D, RAB3D, member RAS oncogene family; RAB3D.L, RAB3D L homeolog; RAB3D.S, RAB3D S homeolog; RTPK, receptor-type tyrosine/protein phosphatase k; THIBZ.S, thyroid hormone induced bZip protein S homeolog; TMEM205, transmembrane protein 205; TMEM205.L, TMEM205 L homeolog; UBAP1, ubiquitin- associated protein 1; UBE2R2, ubiquitin-conjugating enzyme E2 R2.

Induced expression of ISGs, MX1, Viperin, and IFIT-5 by type I the two receptor subunit expression plasmids, pcDNA3.1–Np- IFNs from N. parkeri IFNAR1 and –Np-IFNAR2 together, or separately, with each of To analyze the expression of ISGs induced by type I IFNs from the two receptors. The significant increase was observed in the N. parkeri, and to verify whether these genes were associated with expression of three ISGs in A6 cells following the stimulation by their receptors (Np-IFNAR1 and Np-IFNAR2 to induce the expression each of the three rNp-IFNs, and the expression level of ISGs in- . of ISGs) the three type I IFN expression plasmids, pcDNA3.1–Np- duced by rNp-IFN1 ( 25-fold) was much higher than that of , IFN1, –Np-IFNi1, and –Np-IFNi2 were transfected separately into rNp-IFNi1 and rNp-IFNi2 ( 10-fold; Supplemental Fig. 3). The A6 cells in combination with the two receptor subunit expression expression of ISGs induced by each of the three rNp-IFNs was plasmids, pcDNA3.1–Np-IFNAR1 and –Np-IFNAR2, or each of the also further elevated with the cotransfection of Np-IFNAR1 and two plasmids. In general, the single transfection of each of the three Np-IFNAR2 (Supplemental Fig. 3). IFN expression plasmids, or in combination with the single or the two receptor subunits, all induced significantly the expression of Antiviral activity of type I IFNs from N. parkeri against the three ISGs, including MX1, Viperin, and IFIT-5 (Fig. 6A–C). For FV3 infection the single transfection of each of the three IFN expression plasmids, To examine the antiviral effect of three type I IFNs from N. parkeri, a higher level in the expression of three ISGs was observed for A6 cells were transfected separately with the expression plasmids Np-IFN1 (.30-fold; Fig. 6A), than for Np-IFNil and Np-IFNi2 of these IFNs in combination with single or two receptor subunits (,10-fold; Fig. 6B, 6C). Notably, with the cotransfection of Np- (Fig. 8), as performed above. Transfected A6 cells were then in- IFNAR1 and Np-IFNAR2, the expression level of ISGs induced fected with FV3, and 3 d postinfection, CPE was markedly ob- by the three IFNs was significantly higher than other transfection served in control, which was transfected with the empty plasmid combinations (Fig. 6A–C). pcDNA3.1 (Fig. 7A). However, no CPE was observed in A6 cells Using rNp-IFNs, a similar pattern in the expression of MX1, transfected with Np-IFN1 only and also in combination with the Viperin, and IFIT-5 was also observed in A6 cells transfected with receptors (Fig. 7B). The transfection of Np-IFNi1 or Np-IFNi2 in 8 TIBETAN FROG TYPE I IFNs

combination with the two receptor plasmids together also resulted in a similar protective effect (Fig. 7C, 7D), as observed for Np-IFN1. The transfection of Np-IFNi1 or Np-IFNi2 alone, or in combination with each of the two receptor subunits, had a relatively weak protective effect (Fig. 7C, 7D). As shown in Fig. 7E, each of the three IFNs, Np-IFN1, Np-IFNi1, and Np-IFNi2, when transfected alone or with one or two receptor subunits in A6 cells, caused the significant reduction in FV3 burden compared with that in FV3-infected empty vector-transfected A6 cells. Consistent with the CPE observation, the transfection of A6 cells with Np-IFN1 had a significant and stronger inhibitory effect on virus titer than other two IFNs, Np-IFNi1 or Np-IFNi2, with either Np-IFNAR1 or Np-IFNAR2 expression plasmids or the two together (Fig. 7E). The transfection of Np-IFNi1 or Np-IFNi2 in combination with the two receptor plasmids together in A6 cells had significant and similar inhibitory effect as observed in Np- IFN1–transfected A6 cells. Other combinations of plasmids expressing Np-IFNi1 or Np-IFNi2 with each of the two receptor

subunits resulted in relatively weak inhibitory effects (Fig. 7E). Downloaded from

Discussion Recently, it has been revealed that intron-containing and intronless type I IFN genes coexist in the amphibian species model Xenopus spp. (21, 22). However, the evolutionary and phylogenetic rela-

tionship in intronless type I IFNs between amphibians and amniotes http://www.jimmunol.org/ remains controversial, although they share same genomic structure (20–22). It is imperative to ﬁnd new evidence from other amphibian species to judge whether intronless type I IFNs in amphibians are the ancestral genes of type I IFNs in amniotes or just represent the independent bifurcation in the amphibian lineage. In the current study, the identiﬁcation of intron-containing and intronless type I IFN genes in the Tibetan frog, together with intronless type I IFNs in Xenopus reported previously (21, 22), provides four aspects of evidence to support the hypothesis that by guest on September 28, 2021 intronless type I IFN genes in N. parkeri and Xenopus may have arisen from two independent retroposition events after the divergence of amphibians and amniotes. First, as revealed in sequence homology analyses, there exists a much higher degree of sequence difference between intronless and intron-containing type I IFNs in Xenopus than those in N. parkeri, which may be resulted from the retroposition event in Xenopus that occurred much earlier than the one in N. parkeri. Second, consistently, a relatively closer phylogenetic relationship between intronless and intron-containing type I IFNs in N. parkeri than the counterparts in Xenopus may also support the independent divergence event in these two animals. Third, the different loci where intronless type I IFNs in Xenopus and N. parkeri, and in amniotes, are distributed may provide syntenic evidence to assume that three independent retroposition events might have occurred in Xenopus, N. parkeri, and amniotes, giving rise to the occurrence of intronless type I IFN genes in these species. Owing to the degree of randomness when retrocopies of parental genes were inserted into new genomic regions (17), intronless type I IFN genes in Xenopus, N. parkeri and amniotes are located in different genomic positions.

Table I. Genes and species investigated in this study are marked in red. Ac, green anole lizard A. carolinensis; Am, Mexican axolotl A. mexicanum; FIGURE 3. Phylogenetic tree of type I IFNs in vertebrates constructed by CI, grass carp Ctenopharyngodon idellus; Cp, painted turtle C. picta; using BI method. Branch support values when $0.5 were shown on the tree. Dr, zebraﬁsh D. rerio; Gg, chicken G. gallus; Hs, human H. sapiens; Mm, Type I IFNs from different species are abbreviated accordingly. Sequences mouse M. musculus; Np, Tibetan frog N. parkeri; Xl, African clawed frog used for the phylogenetic tree construction are given in Supplemental X. laevis; Xt, tropical clawed frog X. tropicalis. The Journal of Immunology 9

FIGURE 4. Estimation of divergence times (as MYA) between intron-containing and intronless type I IFN genes in N. parkeri and Xenopus by using RelTime method. Nucleotide coding sequences of type I IFN genes from Tibetan frog (N. parkeri [Np]), tropical Downloaded from clawed frog (X. tropicalis [Xt]), African clawed frog (X. laevis [Xl]), human (H. sapiens [Hs]), mouse (M. musculus [Mm]), chicken (G. gallus [Gg]), green anole lizard (A. carolinensis [Ac]), zebraﬁsh (D. rerio [Dr]), and basking

shark (C. maximus [Cm]) were aligned http://www.jimmunol.org/ by structure-guided alignment method, and nucleotide sequences of these type I IFN genes were aligned according to the results of structure-guided amino acid sequence alignments. All positions containing gaps and missing data in nucleotide sequence alignments were eliminated, and the ML tree was constructed with the Tamura–Nei model of nucleotide substitutions. The TimeTree by guest on September 28, 2021 was computed using three calibration constraints. One calibration was based on the assumption that teleosts and tetrapods were diverged at ∼425–446 MYA, as reported previously (39), secondary calibration date referred to the divergence of reptiles and mammals was at ∼297–326 MYA (39, 40), and third calibration date referred to N. parkeri and Xenopus at ∼187–220 MYA (39, 41). Type I IFN gene from basking shark was used as outgroup. Sequences used for divergence time estimation are given in Supplemental Table I.

The last, and probably the most direct, evidence to support this and Xenopus. The divergence time of intron-containing and hypothesis is derived from the estimation of divergence times of intronless type I IFN genes is 87.57 MYA in N. parkeri and intron-containing and intronless type I IFN genes in N. parkeri 180.70 MYA in Xenopus, respectively, implying that different 10 TIBETAN FROG TYPE I IFNs Downloaded from http://www.jimmunol.org/

FIGURE 5. The expression of type I IFN genes in different organs/tissues of healthy N. parkeri and those treated with poly(I:C). (A) The expression of type I IFN genes in different organs/tissues of healthy N. parkeri (n = 3). (B) The expression of type I IFN genes in different organs/tissues of N. parkeri (n = 3) treated with poly(I:C). Injection of amphibian PBS (APBS) served as control. Gene expression was measured by quantitative RT-PCR, and each sample was run with three technical replicates. The expression data were normalized against the level of b-actin, and fold changes were calculated relative by guest on September 28, 2021 to control group. Data were expressed as mean 6 SE. *p , 0.05. retroposition events might have occurred in these two animals events might have occurred much later than the divergence be- for the presence of intronless type I IFNs in N. parkeri and in tween amphibians and amniotes. It can then be proposed that the Xenopus, and the occurrence of intronless type I IFN genes in retrotransposition for the generation of intronless type I IFN N. parkeri is a much more recent evolutionary event than the genes in amniotes is the third independent event beyond the two occurrence of such genes in Xenopus. In contrast, it has been in amphibians. Intronless type I IFNs in N. parkeri and Xenopus reported that the divergence time for the Amphibia/Amniota may not be the ancestral genes of intronless type I IFNs in separation is ∼350 MYA (59–61), providing equally important amniotes, but may be resulted from two independent retro- evidence to suggest that these two independent retroposition position events in amphibian lineage (Fig. 8).

FIGURE 6. Induction of ISGs in A6 cells following the transfection of expressing plasmids of Np-IFN1 (A), Np-IFNi1 (B), and Np-IFNi2 (C). A6 cells were cotransfected separately with empty pcDNA3.1 vector (as control), expressing plasmids of each of the three Np-IFNs, or together with different combinations of pcDNA3.1–Np-IFNAR1 and –Np-IFNAR2. Then, the cells were harvested, and RNA was extracted to detect the expression of ISGs including MX1, Viperin, and IFIT-5. The experiments were conducted three times, each with three technical replicates. The expression of target genes was normalized against the level of b-actin, and fold changes were calculated relative to control group. Data were expressed as mean 6 SE. *p , 0.05. The Journal of Immunology 11

FIGURE 7. Antiviral activity of type I IFNs from N. parkeri against FV3 in A6 cells. A6 cells were cotransfected separately with empty pcDNA3.1 vector (as control; A), or with expressing plasmids of each of the three Np-IFNs, Np-IFN1 (B), Np-IFNi1 (C), and Np-IFNi2 (D), or together with different combinations of pcDNA3.1–Np-IFNAR1 and –Np- IFNAR2, followed by FV3 infection at multiplicity of infection of 0.3 for 72 h. Three days postinfection, the culture supernatants were removed, and the cell monolayers were ﬁxed with 10% paraformaldehyde Downloaded from and stained with 0.5% crystal violet. (E) Transfection of A6 cells with expressing plasmids of each of the three Np-IFNs reduces the viral yields of FV3. Three days postinfection, A6 cells were subjected to three rounds of sequential freeze/thaw lysis, and the resulting homogenates were collected for the determination of virus titer by standard plaque assays on http://www.jimmunol.org/ EPC cells. The experiments were conducted three times, each with three technical replicates. Data were expressed as mean 6 SE. *p , 0.05. by guest on September 28, 2021

It is a bit surprising that type I IFNs in N. parkeri and Xenopus complexcomposedofIFNAR1andIFNAR2(7,8).UnlikeIFNAR1 were expressed in different patterns and exhibited different antiviral in mammals and birds, which contains four FNIII domains in the activities. As revealed in the current study, both intronless and in- extracellular region (7, 8), the homolog of IFNAR1 in fish, CRFB5, tron-containing type I IFNs in N. parkeri were expressed at very has only two FNIII domains (54–56). Interestingly, the extracellular low levels in unstimulated organs/tissues, but were highly induced region of Np-IFNAR1 contains only two FNIII domains, being similar by poly(I:C) stimulation, and they showed strong ability to induce to CRFB5 in fish. Given that the accretion of pre-existing domains ISGs and strong antiviral effect, as observed in most type I IFNs in appears to have profound impacts on the evolution of cytokine re- mammals as well as in fish (1, 3, 13, 20, 62). However, previous ceptors (65), it is reasonable to speculate that the four FNIII domain– research has shown that intron-containing type I IFNs in Xenopus containing IFNAR1 genes in warm-blooded vertebrates may have were typical inducible cytokines with strong antiviral activity, arisen from primitive two FNIII domain–containing IFNAR1 genes whereas most intronless type I IFNs in Xenopus were constitutively via a tandem domain duplication event in a transit period from cold- expressed at basal expression level but were poorly inducible fol- blooded to warm-blooded vertebrates. The identification of type I IFN lowing poly(I:C) stimulation or virus infection and had very weak receptors in reptiles and other amphibians may provide convincing activity against virus infection, at least in A6 cells (21, 22, 26, 63, evidence to verify the hypothesis. 64). Therefore, it seems possible that intronless and intron-con- It is important to note that amphibians all around the world are taining type I IFNs in N. parkeri are functional with high degree of experiencing dramatic decline in their population sizes and even similarity in terms of the expression and bioactivity, but intronless local extinctions since the 1980s (66, 67). It is generally accepted and intron-containing type I IFNs in Xenopus may exhibit certain that many catastrophic die-offs of amphibian populations are at- degree of difference in their expression and effect. Notably, tributed to emerging infectious diseases caused by ranaviruses intronless and intron-containing type I IFNs in N. parkeri had ISG- (24, 25). To prevent the outbreak of such diseases has become a inducing and antiviral activities in A6 cells even without the major task for the conservation of some amphibians, especially cotransfection of Np-IFNAR1 and Np-IFNAR2, implying that a those endangered species, and the understanding of their immune certain degree of cross-species reactivity exists between type I IFNs system, particularly their IFN system, may provide clues for the and their receptors at least in different species of anurans. development of strategies on the prevention of such diseases. The initiation of type I IFN–mediated signaling pathway depends on Previously, intron-containing type I IFN and type III IFN in the interaction between type I IFNs and their heterodimeric receptor X. laevis have been reported to possess prominent antiranavirus 12 TIBETAN FROG TYPE I IFNs Downloaded from

FIGURE 8. A revised model for the evolution of type I IFNs in vertebrates. Fish may possess the most primitive type I IFNs with a five-exon and four- intron gene organization. During a transition period when vertebrates migrated from aquatic to terrestrial environments, at least two retroposition events might have occurred in amphibians, at 180.70 MYA in Xenopus and 87.57 MYA in N. parkeri, thus leading to the generation of intronless type I IFN genes in N. parkeri and Xenopus, respectively, which may represent two independent bifurcations in the amphibian lineage. These two independent retroposition http://www.jimmunol.org/ events might have occurred much later than the divergence between amphibians and amniotes, which might have occurred at ∼350 MYA (red dot) (59–61). It can then be proposed that intronless type I IFN genes in amniotes may have arisen from another retroposition event that occurred in a transition period when reptiles were diverged from amphibians. Intron-containing type I IFN genes should have been lost in reptiles, and so in all amniotes, including reptiles, birds, and mammals, which possess only intronless type I IFNs. Retroposition event, a RNA-based duplication mechanism which creates duplicate genes in new genomic positions through the reverse transcription of expressed parental genes, is indicated with an arrow. effect (26, 46, 63, 64). In the current study, the identified intronless adjacent to FOCAD and HACD4 in birds, as seen in mammalian type I IFNs, Np-IFNi1 and Np-IFNi2, in the Tibetan frog have IFN-a and IFN-b. The evolution of this predicted type I IFN gene been proven to have antiviral activity against FV3, implying that needs to be investigated when its predicted cDNA sequence can be by guest on September 28, 2021 intronless type I IFN in amphibians may also play important roles confirmed in vivo. By contrast, the IFN-a and IFN-b genes in in immune response against ranaviruses. chicken are linked to distinct genes, including NOL6, UBE2R2, Unlike the complicated type I IFN system in Xenopus, which DCAF12, and UBAP1. Interestingly, all type I IFN genes in contains three groups of intronless type I IFNs and two groups of chicken are located at chromosome Z, so the IFN-a and IFN-b intron-containing type I IFNs (21, 22), only three type I IFN genes gene in chicken may be resulted from an intrachromosomal gene have been identified in the N. parkeri genome. In addition, Np- duplication event that occurred independently in birds. Consis- IFNi–f1 and Np-IFNi–f2, linked to Np-IFNi1, were found in the tently, IFN-a in birds and mammals, as well as IFN-b in birds and current study, but these two fragments appear undetectable in vivo mammals, were not grouped together in phylogenetic tree. The at the transcription level. Despite the presence of unsequenced IFN-a and IFN-b in birds were grouped together to form a sep- gaps in the genome, it is evident that N. parkeri contains fewer arate clade beyond the clade containing type I IFNs in mammals. type I IFN members when compared with Xenopus (21, 22), which Much more obviously, the divergence time of IFN-a and IFN-b, may be an important factor related with the difference in sus- which is 252.09 MYA in mammals, but 36.76 MYA in birds, may ceptibility to ranavirus infection in different amphibian species. suggest the divergence of avian IFN-a and IFN-b is a much recent N. parkeri belongs to the suborder Neobatrachia which contains evolutionary event. Therefore, it may be necessary to revise the ∼85% of the known amphibian species, and species in the sub- nomenclature of avian type I IFNs to represent truly their evolu- order appears to be more susceptible to ranavirus infection tionary origin (14). (23, 68). In contrast, Xenopus, belonging to Archaeobatrachia, Based on the current knowledge of type I IFNs in vertebrates and show strong resistance to ranavirus and may serve as a potential the findings in the current study, a revised model can be proposed vector for ranaviruses (45, 69). It is reported that type I IFNs are in with a certain degree of confidence to explain the possible evo- tight association with the resistance against viruses (1, 4, 44), and lutionary route of type I IFNs in vertebrates (Fig. 8). It is con- the discrepancy in the composition of type I IFN system between sidered that fish may possess the most primitive type I IFNs with a neobatrachian species and Xenopus may account for their differ- five-exon and four-intron gene organization. During a transition ence in the susceptibility or resistance to ranavirus infection. The period when vertebrates migrated from aquatic to terrestrial en- characterization of type I IFN system in other neobatrachian vironments, at least two retroposition events might have taken species may be of interest for further research. place in amphibians, thus leading to the generation of intronless Previous studies about the avian type I IFNs have indicated that type I IFN genes in Xenopus and N. parkeri, respectively. But the avian IFN-a and IFN-b may not be the true orthologs of mam- intronless type I IFN genes in these two lineages are not the direct malian IFN-a and IFN-b, respectively (14, 70), which is further orthologs of type I IFN genes in amniotes, and may just represent supported by the evolutionary evidence revealed in the current two independent bifurcations in the amphibian lineage. Intronless study. First, only one predicted type I IFN gene, intronless, is type I IFN genes in amniotes may have arisen from another The Journal of Immunology 13 retroposition event that occurred in a transition period when rep- Tibetan frog Nanorana parkeri and the comparative evolution of tetrapod ge- nomes. Proc. Natl. Acad. Sci. USA 112: E1257–E1262. tiles were diverged from amphibians. Then, intron-containing type 24. Robert, J. 2010. Emerging ranaviral infectious diseases and amphibian decline. I IFN genes have been lost in reptiles; so all classes of amniotes, Diversity 2: 314–330. including reptiles, birds, and mammals, possess only intronless 25. Price, S. J., T. W. Garner, R. A. Nichols, F. Balloux, C. Ayres, A. Mora-Cabello de Alba, and J. Bosch. 2014. Collapse of amphibian communities due to an type I IFNs. Future genome sequencing of primitive reptiles may introduced Ranavirus. Curr. Biol. 24: 2586–2591. help to understand the retroposition event for the occurrence of 26. Grayfer, L., F. De Jesu´s Andino, and J. Robert. 2014. The amphibian (Xenopus intronless type I IFNs in amniotes. laevis) type I interferon response to frog virus 3: new insight into ranavirus pathogenicity. J. Virol. 88: 5766–5777. 27. Larkin, M. A., G. Blackshields, N. P. Brown, R. Chenna, P. A. McGettigan, Acknowledgments H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, R. Lopez, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947–2948. We thank all the laboratory members for critical reviews and comments on 28. Hamming, O. J., G. Lutfalla, J. P. Levraud, and R. Hartmann. 2011. Crystal this manuscript, and our special gratitude is devoted to Prof. Jing Che at the structure of Zebrafish interferons I and II reveals conservation of type I inter- Kunming Institute of Zoology, Chinese Academy of Sciences for the field feron structure in vertebrates. J. Virol. 85: 8181–8187. sample arrangement. 29. Kececioglu, J., E. Kim, and T. Wheeler. 2010. 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Species Name GenBank accession no Species Protein GenBank accession no H. sapiens Hs-IFN-α1 NP_076918.1 G. gallus Gg-IFN-α7 XP_004937149 Hs-IFN-α2 EAW58611.1 Gg-IFN-α8 XP_015132923 Hs-IFN-α4 NP_066546.1 Gg-IFN-α9 XP_004937147 Hs-IFN-α5 NP_002160.1 Gg-IFN-α10 XP_015132924 Hs-IFN-α6 NP_066282.1 Gg-IFN-α11 XP_015132926 Hs-IFN-α7 NP_066401.2 Gg-IFN-β NP_001020007 Hs-IFN-α8 NP_002161.2 Gg-IFNAR1 NP_990190.1 Hs-IFN-α10 NP_002162.1 Gg-IFNAR2 NP_990189.1 Hs-IFN-α13 NP_008831.3 A. carolinensis Ac-IFN1–3 XP_016846269.1–XP_008101701.1 Hs-IFN-α14 NP_002163.2 Ac-IFN4 XP_008101702.1 Hs-IFN-α16 NP_002164.1 C. picta Cp-IFNi4 XP_023961202.1 Hs-IFN-α17 NP_067091.1 Cp-IFNi5 XP_005297844.1 Hs-IFN-α21 NP_002166.2 Cp-IFNi7 XP_005297843.1 Hs-IFN-β EAW58625.1 X. tropicalis Xt-IFN1–4 BN001167.1–BN001170.1 Hs-IL-10 NP_000563.1 Xt-IFN5 KX592539 Hs-IFNAR1 CAA42992.1 Xt-IFNi1.1 MF356931 Hs-IFNAR2 CAA61914.1 Xt-IFNi1.2 KX592540 M. musculus Mm-IFN-α1 NP_034632.2 Xt-IFNi1.3–1.7 MF356932–MF356936 Mm-IFN-α2 NP_034633.2 Xt-IFNi2.1 KX592541 Mm-IFN-α4 NP_034634.1 Xt-IFNi2.2–2.4 MF356937–MF356939 Mm-IFN-α5 NP_034635.2 Xt-IFNi3.1 KX592542 Mm-IFN-α6 NP_996754.1 Xt-IFNi3.2–3.5 MF356940–MF356943 Mm-IFN-α7 NP_032360.2 X. laevis Xl-IFN1 KU594561 Mm-IFN-α9 NP_034637.1 Xl-IFN3 KU594563 Mm-IFN-α11 NP_032359.2 Xl-IFN4 KU594564 Mm-IFN-α12 NP_796335.1 Xl-IFN7 KU594567 Mm-IFN-α13 NP_796321.1 Xl-IFNX4–5 KU594571–KU594572 Mm-IFN-α14 NP_996858.1 Xl-IFNX7 KU594574 Mm-IFN-α15 NP_996753.1 Xl-IFNX9 KU594576 Mm-IFN-α16 NP_996750.1 Xl-IFNX12–13 KU594579–KU594580 Mm-IFN-β NP_034640.1 Xl-IFNX15 KU594582 Mm-IFNAR1 NP_034638.2 Xl-IFNX20 KU594587 Mm-IFNAR2 NP_034639.2 D .rerio Dr-IFN1 NM_207640.1 G. gallus Gg-IFN-α1 XP_004937153 Dr-IFN2–3 NM_001111082.1–NM_001111083.1 Gg-IFN-α2 XP_004937152 Dr-CRFB1 NP_001073149.1 Gg-IFN-α3 XP_004937151 Dr-CRFB2 NP_001071094.2 Gg-IFN-α4 XP_015132925 Dr-CRFB5 NP_001029357.3 Gg-IFN-α5 XP_004937150 C. idella CI-IFN2–3 AMT92190.1–AMT92191.1 Gg-IFN-α6 NP_990758 C. maximus Cm-IFN XM_007885692.1

Supplemental Figure 1. Multiple alignments of deduced type I IFN receptor protein sequences in N. parkeri . Identical amino acids are indicated with “*”, and amino acids with high and low similarity are indicated with “:” and “.”, respectively. The putative signal peptides and cysteine residues participated in formation of disulfide bonds are underlined and in bold, respectively. The amino acid sequences from different species are named accordingly and abbreviated as: Np, Tibetan frog N. parkeri ; Xt, tropical clawed frog X. tropicalis ; Hs, human H. sapiens ; Mm, mouse M. musculus ; Gg, chicken G. gallus ; Dr, zebrafish D. rerio . (A) Multiple alignments of deduced IFNAR1 protein sequence in N. parkeri . The putative FNIII domains of Np-IFNAR1 and Dr-CRFB5 are highlighted in solid lines above the alignment, and corresponding sequences are illustrated in light grey shadow. Deep grey shadow sequences indicate predicted transmembrane regions of IFNAR1. The DSGXY motif that is vital for ubiquitination, endocytosis and degradation of IFNAR1 is in bold. ( B) Multiple alignments of deduced IFNAR2 protein sequence in N. parkeri . The putative FNIII domains of Np-IFNAR2, Dr-CRFB1 and Dr-CRFB2 are highlighted in solid lines above the alignment, and corresponding sequences are illustrated in light grey shadow. Deep grey shadow sequences indicate predicted transmembrane regions of IFNAR2. The tyrosine residue which plays a critical role in activation of STAT1 and STAT2 is in bold.

Supplemental Figure 2. Phylogenetic tree of type I IFNs in vertebrates constructed by using maximum likelihood ( A) and neighbor-joining (B) methods. Branch support values when ≥ 50 were shown on the tree. Type I IFNs from different species are abbreviated accordingly as: Np, Tibetan frog N. parkeri ; Xt, tropical clawed frog X. tropicalis ; Xl, African clawed frog X. laevis ; Hs, human H. sapiens ; Mm, mouse M. musculus ; Am, Mexican axolotl A. mexicanum ; Gg, chicken G. gallus ; Ac, green anole lizard A. carolinensis ; Cp, painted turtle C. picta ; Dr, zebrafish D. rerio ; CI, grass carp C. idellus .

Supplemental Figure 3. Induction of ISGs, MX1, Viperin, IFIT-5 in A6 cells following the stimulation of recombinant Np-IFNs, rNp-IFN1 ( A), rNp-IFNi1 ( B), rNp-IFNi2 ( C), with the rNp-IFNs detected by Western blotting (D). The rNp-IFNs were collected in supernatants of transfected HEK293T cells. The pcDNA3.1 vectors carrying full-length coding sequences of Np-IFNs with HA-tag sequence in 3'-end region were transfected into HEK293T cells. 48 hours post-transfection, Np-IFNs in medium were confirmed by Western blotting using anti-HA-tag antibody. A6 cells were co-transfected with different combinations of pcDNA3.1-Np-IFNAR1 and -Np-IFNAR2, and then the cells were incubated respectively for 10 h with 200 µl supernatant of transfected HEK293T cells containing rNp-IFN1, rNp-IFNi1 or rNp-IFNi2, and an equal volume of the vector control. The cells were harvested and RNA was extracted to detect the expression of ISGs. The experiments were performed for three biological replicates with three technical replicates for each biological replicate. The expression of target genes was normalized against the level of β-actin, and fold changes were calculated relative to control group. Data were expressed as mean ± SE, with * indicating P < 0.05.