Molecular characterization of woodchuck IFI16 and AIM2 and their expression in woodchucks infected with woodchuck hepatitis virus (WHV)

IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2, and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-β were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1β were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-β stimulated by VACV ds 70 mer, and AIM2 siRNA interference downregulated AIM2 and IL-1β transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-β and IL-1β, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.

Although the chimpanzee is the only animal that can be infected by HBV, the use of this animal is expensive and ethically limited 27 . Woodchuck hepatitis virus (WHV) is a member of the hepadnaviridae family and can infect the American woodchuck (M. monax) and the Chinese woodchuck (M. himalayana). Woodchuck WHV infection strongly resembles human HBV infection in its major virological, pathological, and immunological features 1,[28][29][30] . Hence, the woodchuck model is valuable for studying the pathogenesis of HBV infection, evaluating antiviral drugs and developing rational immunotherapeutic strategies 27,29,31,32 . However, the intracellular innate DNA sensors IFI16 and AIM2 require further study in the woodchuck model.
In this study, we characterized the complete coding sequences (CDSs) of woodchuck IFI16 (wIFI16) and AIM2 (wAIM2), examined their roles in IFN-β and IL-1β induction in woodchuck fibroblastoma cells, respectively, and evaluated the effect of their respective ligands, VACV ds 70 mer and poly (dA:dT), in vivo. Additionally, we measured the expression of wIFI16 and wAIM2 transcripts in different organs from naïve woodchucks and in the liver and spleen of woodchucks with different WHV-infected status.

Results
Sequence analysis of wIFI16 and wAIM2. The CDS of wIFI16 was submitted to GenBank (accession number KP334127) and consists of 2574 bp with a deduced protein length of 857 amino acids (aas). The sequence identities between wIFI16 and its orthologues in other mammalian species ranged from 52.53 to 76.26% and from 29.27 to 65.33% at the nucleotide (nt) and aa levels, respectively (Table 1). A phylogenetic tree was constructed using the sequences of IFI16 from woodchuck and other mammalian species, indicating that wIFI16 was closely related to mouse IFI16 (Fig. 1A). The Conserved Domain Search Service (CD Search) indicated that wIFI16 contains an N-terminal Pyrin domain (10-83aa) and a C-terminal DNA-binding HIN domain (647-815aa), and the MFHATVAT motif was conserved in the HIN domain ( Fig. 2A) 33 . The alignment analysis found 2 cysteine residues (Cys801 and Cys811) that were conserved in the HIN domain of woodchucks and other mammalian species ( Fig. 2A).
The CDS of wAIM2 was submitted to GenBank (accession number KP272148) and is 1050 bp in length. The deduced protein has 349 aa residues. The sequence of wAIM2 exhibited high sequence identity with its orthologues from other mammalian species, ranging from 63.58 to 78.14% at the nt level and from 52.80 to 70.09% at the aa level ( Table 2). The phylogenetic tree showed that wAIM2 is closest to its human and chimpanzee orthologues (Fig. 1B). The CD Search indicated that there were two conserved domains, the N-terminal Pyrin domain (10-82aa) and the C-terminal DNA-binding HIN domain (157-326aa) in wAIM2, and the M(I)FHATVAT and Rb-binding motifs were conserved (Fig. 2B). A previous study indicated that mutating Lys160, Lys204, Lys251, Lys309, Phe165 or Arg244 in the HIN domain of human AIM2 impairs its binding activity with dsDNA 34 . Accordingly, all of the corresponding sites in the HIN domain of wAIM2 were conserved (Fig. 2B).
Silencing wIFI16 or wAIM2 impaired IFN-β or IL-1β transcript expression, respectively, in woodchuck fibroblastoma cells. The intracellular VACV ds 70 mer and poly (dA:dT) are known ligands of IFI16 and AIM2, respectively, and can induce IFN-β and IL-1β production, respectively, in human and mice 17,21 . To determine whether these two dsDNAs can induce IFN-β and IL-1β production in woodchuck cells, respectively, we examined their mRNA levels in a woodchuck fibroblastoma cell line (WH12/6) with or without VACV ds 70 mer or poly (dA:dT) stimulation. The transcripts of wIFI16 and wIFN-β were dramatically increased, whereas wRelA (a subunit of NF-κ B) and wIRF3 were only slightly increased, in VACV ds 70 mer-transfected cells compared with those of the VACV ss 70 mer-transfected or mock-infected cells (Fig. 3A,B). Similarly, wAIM2 and wIL-1β transcripts were upregulated after poly (dA:dT) transfection in WH12/6 cells (Fig. 3C). These data indicate that, at the mRNA level, human and mouse IFI16 and AIM2 ligands can stimulate wIFI16 and wAIM2, respectively, and lead to wIFN-β and wIL-1β production, respectively.

Species
Common name Accession no. Similarly, to determine whether poly (dA:dT)-induced wIL-1β production was mediated by wAIM2, we measured wAIM2 and wIL-1β production after wAIM2-targeted small interfering RNA (siRNA) interference. Six siRNAs targeting wIFI16 or wAIM2 (three each) were synthesized, and the siRNAs that reduced gene target expression by more than half were used in the subsequent silencing experiments on mRNA levels ( Supplementary  Fig. S1). The siRNA targeting wIFI16 (siIFI16) caused a comparable reduction in wIFI16 mRNA expression in VACV ds 70 mer-transfected and mock-infected cells (Fig. 3D). As expected, wIFN-β mRNA expression was partially inhibited in siIFI16-transfected cells with or without VACV ds 70 mer stimulation (Fig. 3D), suggesting another DNA sensor may be involved in VACV ds 70 mer-mediated IFN-β induction in woodchucks. A previous study showed that both IFI16 and the DEAD-box helicase 41 (DDX41) are responsible for VACV DNA mediated IFN-β induction in a human monocytic cell line (THP-1) 35 , while whether DDX41 or other DNA sensors are involved in woodchuck fibroblastoma cells requires further investigation. SiIFI16 inhibited wRelA and wIRF3 mRNA expression in VACV ds 70 mer-transfected cells but not in the mock-infected control cells (Fig. 3E). The siRNA targeting wAIM2 (siAIM2) decreased wAIM2 mRNA expression in both poly (dA:dT)-transfected and mock-infected cells (Fig. 3F) and blocked IL-1β mRNA production with or without poly (dA:dT) stimulation.

IFN-β and IL-1β induction by the respective ligands of wIFI16 and wAIM2 in vivo.
To evaluate the effect of the respective ligands of IFI16 and wAIM2 in vivo, we delivered VACV ds 70 mer and poly (dA:dT) into woodchucks with the Entranster ™ -in vivo transfection reagent and measured the mRNA levels of wIFI16, wAIM2 and their respective effectors, wIFN-β and wIL-1β in peripheral blood mononuclear cells (PBMCs) and the liver. After VACV ds 70 mer in vivo transfection, wIFI16 and wIFN-β transcript levels increased to a maximum on day 1 in PBMCs and were upregulated on day 3 in the liver (Fig. 4A,C). After poly (dA:dT) in vivo transfection, wAIM2 and wIL-1β transcripts reached a peak on day 1 in PBMCs and moderately increased on day 3 in the liver (Fig. 4B,D), indicating that VACV ds 70 mer and poly (dA:dT) could induce the transcription of IFI16, wAIM2 and their respective effectors, wIFN-β and wIL-1β , in vivo.
Basal wIFI16 and wAIM2 expression analysis. The samples from two naïve woodchucks were collected to investigate the transcriptional levels of wIFI16 and wAIM2 in different organs (Fig. 5). The expression of wIFI16 transcripts was highest in the kidney, followed by the stomach, liver, lung, spleen and intestine. Low-level  wIFI16 expression was found in the brain, heart, muscle and testis. The expression of wAIM2 was abundant in the stomach, followed by the brain, thymus and spleen, whereas low-level wAIM2 expression was observed in the lung, liver, testis and heart. wAIM2 transcripts were not detected in the kidney.
The expression of wIFI16 and wAIM2 in liver and spleen tissues from WHV-infected woodchucks. Twenty woodchucks were used in this study, consisting of 6 naïve (healthy) woodchucks, 5 woodchucks acutelyinfected with WHV, 5 resolved animals, and 4 woodchucks with chronic WHV infection. The descriptive statistics are listed in Supplementary Table S1. The expression levels of wIFI16 and its downstream molecule, wIFN-β , were increased in the livers and spleens from the WHV acutely infected animals, and were slightly decreased in the liver from woodchucks chronically infected with WHV, when compared with those from naïve (healthy) and/or resolved woodchucks (Fig. 6). The expression levels of wAIM2 and its downstream molecule, wIL-1β , were increased in the livers of woodchucks with acute and resolved WHV infection, and were increased in the spleens of woodchucks with acute WHV infection compared with the spleens of naïve (healthy) woodchucks (Fig. 6).

Discussion
Experimentally WHV-infected woodchucks provide a valuable animal model for studying host-HBV interactions, but the lack of sequence information hampers functional genomics analyses in this model. To address this major limitation of the model, Fletcher et al. conducted a transcriptome analysis in woodchucks, providing us with a large number of woodchuck gene sequences and a valuable custom woodchuck microarray 36,37 . However, the intracellular innate DNA sensors IFI16 and AIM2 still require further investigation in woodchucks. In the present study, we cloned and analyzed the full-length CDSs of wIFI16 and wAIM2, examined their roles in IFN-β and IL-1β induction in vitro, evaluated the effect of their respective ligands in vivo, and measured their expression in WHV-infected woodchucks. The deduced wIFI16 and wAIM2 proteins had 857 and 349 aa residues, respectively, and their calculated molecular weights were similar to the previously reported sizes of human and mice IFI16 and AIM2 15,21,25 . The sequence identity of wIFI16 with its orthologues from other mammalian species was as low as 30% at the aa level, while wAIM2 was relatively conserved, with a sequence identity higher than 50% at both the nt and aa levels. This result is consistent with a previous study that indicated AIM2, but not the IFI16 sequence, was highly conserved in human and mice 9 . Furthermore, we also found that both wIFI16 and wAIM2 contain the important functional structures of the ALR family, the DNA-binding HIN domain and the adaptor-binding Pyrin domain, which are conserved in human and mice 38,39 . In addition, wIFI16 has conserved cysteine residues (Cys801, Cys811) and a conserved MFHATV motif compared with its orthologues in other species 33,34 , and the M(I)FHATV and Rb-binding motifs were conserved and located at the corresponding sites in wAIM2 38 .
The basal expression of wIFI16 in the woodchuck fibroblastoma cell line was comparatively high, similar to results in human epithelial cells 40 . The VACV ds 70 mer and poly (dA:dT) are known IFI16 and AIM2 ligands, respectively, and were found to induce wIFN-β and wIL-1β expression, respectively, in human and mouse cells 17,[20][21][22][23] . Consistently, wIFI16 and wIFN-β transcripts were upregulated after VACV ds 70 mer transfection in woodchuck cells, while wAIM2 and wIL-1β transcripts were also upregulated after poly (dA:dT) transfection. SiRNA silencing of wIFI16 partially inhibits VACV ds 70 mer-stimulated wIFN-β transcription, and wAIM2 silencing inhibits poly (dA:dT)-induced wIL-1β transcription, indicating that the induction of wIFN-β by VACV ds 70 is partially dependent on wIFI16, while wAIM2 is responsible for poly (dA:dT)-mediated-wIL-1β induction. In addition, we found that wIFI16 and wIFN-β transcripts were upregulated after VACV ds 70 mer in vivo transfection, and wAIM2 and wIL-1β transcripts were also upregulated after poly (dA:dT) in vivo transfection, providing the basis for using wIFI16 and wAIM2 ligands, poly (dA:dT) and VACV ds 70 mer, in the woodchuck model.
The expression of wIFI16 transcripts was abundant in the kidney, stomach, liver, lung and spleen, in which a variety of lymphocytes was colonized as a result of persistent contact with exogenous DNA. Similarly, IFI16 was primarily detected in the lymphoid tissues and in human epithelial cells 40 . A previous study showed that AIM2 plays a critical role in autoimmune diseases of the nervous system and repair 41,42 ; as expected, wAIM2 transcripts were also highly expressed in the woodchuck brain and stomach.
IFI16 and AIM2 play important roles in controlling viral replication 18,19 . Therefore, we investigated the expression of wIFI16, wAIM2 and their downstream molecules, wIFN-β and wIL-1β in the liver (the primary target organ of HBV) and/or the spleen (an important immune organ) of woodchucks infected with WHV. We found

Species
Common name Accession no. that both wIFI16 and its downstream molecule, wIFN-β were upregulated in woodchucks acutely infected with WHV and were slightly downregulated in those with chronic infection. The expressions of wAIM2 and its downstream molecule, wIL-1β were increased in woodchucks with acute and resolved WHV infection but remained almost unchanged in those with chronic infection. This observation is not completely consistent with a previous study in woodchucks. Using microarray analysis, Fletcher et al. found that IFI16 and AIM-2 were increased in the livers of woodchucks with acute and chronic infection, but not in resolved WHV infection 37 . However, our observation is consistent with the findings in humans, in which AIM2 transcript levels were found to be increased in PBMCs from acutely HBV-infected patients but remained nearly unchanged in chronically HBV-infected patients 43 . Taken together, the differential expression pattern of IFI16 and AIM2 transcripts following WHV/ HBV infection implied that these two DNA sensors might be involved in WHV/HBV infection. Further investigation, e.g., using a knockdown strategy, is needed to verify whether IFI16 and AIM2 are essential for controlling WHV/HBV infection.
In conclusion, we identified the DNA sensors IFI16 and AIM2 and their ligands in a woodchuck model, which is a valuable model for studying HBV infection. Our data also imply the possible role of IFI16 and AIM2 in WHV/HBV infection, which is useful for further investigation. These results provide the basis for the further study of IFI16-and AIM2-mediated innate immunity in HBV infection using the informative woodchuck model.

Ethics statement. American woodchucks were purchased from the Institute of Laboratory Animal
Sciences of the Chinese Academy of Medical Sciences and were maintained at the Experimental Animal Center Figure 3. The effects of siIFI16 on IFN-β, and siAIM2 on IL-1β, transcription induction, with or without VACV ds 70 mer or poly (dA:dT) stimulation, respectively. WH12/6 cells were transfected with VACV ds 70 mer (2 μ g/ml) or poly (dA:dT) (2 μ g/ml) for 24 hours. The wIFI16, wIFN-β (A), wRelA, wIRF3 (B), wAIM2, and wIL-1β (C) were quantified using RT-qPCR. The VACV ss 70 mer (2 μ g/ml) was transfected as a control. WH12/6 cells were transfected with siIFI16 or the control siRNAs for 24 hours and then transfected with or without the VACV ds 70 mer (2 μ g/ml). Twenty-four hours later, wIFI16, wIFN-β (D), wRelA and wIRF3 (E) transcripts were quantified using RT-qPCR. WH12/6 cells were transfected with siAIM2 or the control siRNAs for 24 hours and were then transfected with or without poly (dA:dT) (2 μ g/ml). Twenty-four hours later, wAIM2 and wIL-1β (F) transcripts were quantified using RT-qPCR.
Scientific RepoRts | 6:28776 | DOI: 10.1038/srep28776 of Huazhong University of Science and Technology. Chinese woodchucks were captured from Qinghai province, China, and were maintained at the Experimental Animal Center of Qinghai. All efforts were made to alleviate stress and any incidental deaths. The woodchucks were anesthetized via intramuscular ketamine injection. PBMCs were isolated from the blood. Liver and spleen specimens were obtained from the Chinese woodchuck sample pool in our lab. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, revised 1996) and were reviewed and approved by the local Animal Care and Use Committees (Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology, China).
Cloning and the sequence analysis of wIFI16 and wAIM2. Woodchuck PBMCs were isolated and stimulated with 5 μ g/ml concanavalin A (ConA) (Sigma, St. Louis, MO, USA) for 24 hours. Total RNA was extracted using TRIzol (Takara, Dalian, China) according to the manufacturer's instructions and was used as the template for subsequent complementary DNA (cDNA) synthesis. The synthesized cDNA was used as a template for the PCR amplification of the CDSs of wIFI16 and wAIM2. The primers used to generate the wIFI16 and wAIM2 CDSs are listed in Supplementary Table S2. The resulting PCR products were cloned into the pMDT-18 vector according to the manufacturer's instructions (Takara), and the plasmids containing wIFI16 or wAIM2 were sequenced via a commercial service (Invitrogen, Carlsbad, CA, USA).

Woodchuck IFI16 and AIM2 ligands stimulation in vitro and in vivo and siRNA interference.
The WH12/6 cell line was kindly provided by P. Banasch of the German Cancer Research Center, DKFZ Heidelberg, Germany, and maintained in Ham's F12 medium supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco), as described previously 45 .
The VACV ds 70 mer, the VACV ss 70 mer and poly (dA:dT) (InvivoGen, SanDiego, CA, USA) were transfected into WH12/6 cells at a concentration of 2 μ g/ml for 6 hours using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After 18 hours, the cells were harvested, snap-frozen in liquid nitrogen, and then stored at − 80 °C until RNA extraction.
The siRNAs (double-stranded RNA oligonucleotides) and negative control duplexes were chemically synthesized (Invitrogen). The siRNA sequences are listed in Supplementary Table S3. WH12/6 cells were transfected with 100 nM Silencer Select siRNAs for wIFI16 or wAIM2 and the negative control duplexes and were subsequently transfected with the VACV ds 70 mer, the VACV ss 70 mer or poly (dA:dT) at a concentration of 2 μ g/ml using Lipofectamine 2000 (In vitro gen) according to the manufacturer's instructions. After 18 hours, the cells were harvested, snap-frozen in liquid nitrogen, and then stored at − 80 °C until RNA extraction.
The VACV ds 70 mer and poly (dA:dT) (In vivo Gen, SanDiego, CA, USA) were administered into naïve woodchucks (n = 2) at doses of 25 μ g/kg and 50 μ g/kg, respectively, using the Entranster ™ -in vivo transfection reagent (Engreen Biosystem Co. Beijing, China) according to the manufacturer's instructions. PBMCs were isolated from the whole blood at day0, day1, day2 and day3 after ligands administration, while the livers were   collected at day3 after ligands administration, snap-frozen in liquid nitrogen, and then stored at − 80 °C until RNA extraction.
Sample collection, RNA extraction and reverse transcription quantitative real-time PCR (RT-qPCR). Small intestine, large intestine, lung, liver, spleen, kidney, testis, muscle, brain, stomach, heart and thymus specimens from 2 naïve (healthy) woodchucks and liver and spleen samples from 16 woodchucks were collected by necropsy, snap-frozen in liquid nitrogen, and stored at − 80 °C until RNA extraction. Total RNA was extracted from WH12/6 cells and tissue samples using TRIzol (Takara) according to the manufacturer's instruction. The quality and quantity of the RNAs were evaluated by calculating the A260/A280 ratio and 28 S/18 S ribosomal RNA ratio. RT-qPCR was conducted using the RNA templates and the SYBR Green I Kit (Toyobo, Dalian, China) according to the manufacturer's instructions in the CFX Connect ™ Real-Time PCR Detection System (BIO-RAD, Hercules, California, USA). RT-qPCR was carried out in a 20 μ L volume, containing 10 μ L of 2 × Buffer (dNTP mixture, Mg 2 + and SYBR Green I), 0.8 μ L of enzyme mix (RTase, RNase inhibitor and Ex Taq), 0.8 μ L of 10 μ Μ forward primers, 0.8 μ L of 10 μ Μ reverse primers, 2 μ L of 50 ng/μ L total RNA, and 5.6 μ L of RNase free dH 2 O. The cycling parameters were 42 °C for 5 min, 95 °C for 10 s, followed by 40 cycles of 5 s at 95 °C and 30 s at 60 °C. Primer sets were designed using the Primer Express TM 3.0 software (Applied Biosystems, Carlsbad, CA, USA). All these primer pairs produced a single amplicon according to the specific PCR product seen upon 2% agar gel electrophoresis and the single peak dissociation curves of RT-qPCR products. The primers used for the RT-qPCR of wIFI16, wAIM2, wIFN-β , wRelA, wIRF3, wIL-1β and β -actin are listed in Supplementary Table S2. The primer efficiencies were evaluated by the calibration curve generated with serial dilutions (covering 3 orders of magnitude) of cDNA ( Supplementary Fig. S2). The efficiency of all primer pairs ranged from 0.91 to 1.09. The R 2 values (correlation coefficients) were between 0.990 and 0.996. No-template controls were included to ensure the absence of reagent contamination and genomic DNA. All of the reactions were performed in triplicate, and the mean value of the quantification cycle (Cq) was used for subsequent analysis. The quantification was performed using the Pfaffl method 46 by calculating the copy numbers from the Cq-value for each gene per sample. Beta-actin expression was used to normalize the mRNA expression levels of wIFI16, wAIM2, wIFN-β , wRelA, wIRF3 and wIL-1β [47][48][49] , and the expression levels were given as the copy number/100,000 copies of β -actin mRNA.
Statistical analysis. SPSS18.0 (IBM Corporation, Somers, NY, USA) was used to assess the statistical significance of differences (p-values) between the groups using unpaired t-tests with equal variance. A p-value < 0.05 was considered significant.