Length-dependent innate antiviral effects of double-stranded RNA in the rainbow trout (Oncorhynchus mykiss) cell line, RTG-2

Fish & Shellfish Immunology 46 (2015) 557e565

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Length-dependent innate antiviral effects of double-stranded RNA in the rainbow trout (Oncorhynchus mykiss) cell line, RTG-2 Sarah J. Poynter, Stephanie J. DeWitte-Orr* Department of Biology, Wilfrid Laurier University, 75 University Ave W, Waterloo, Ontario N2L 3C5, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 April 2015 Received in revised form 24 June 2015 Accepted 19 July 2015 Available online 21 July 2015

Effectively all viruses produce long dsRNA during their replicative cycle. In mammals long dsRNA molecules induce a robust response through the production of type 1 interferon, interferon-stimulated genes (ISGs) and an antiviral state. This response is less well understood in fish. We investigated the ability of a rainbow trout cell line, RTG-2, to respond to two different lengths of in vitro transcribed dsRNA (200 bp and 1264 bp) based on the viral hemorrhagic septicemia virus genomic sequence, and high and low molecular weight poly I:C (synthetic dsRNA). To explore the innate immune response we used qRT-PCR to measure immune gene transcript levels, an ISG-promoter reporter assay, and an antiviral protection assay. We saw a significantly greater immune response in all assays in response to the longer dsRNA molecule compared to their shorter counterpart. We saw significantly more interferon and ISG transcripts, stronger induction of a protective antiviral state, and more robust activation of the ISG-promoter. This response was not found to be due to a better uptake of the longer dsRNA molecules as a cellular uptake assay showed no differences between lengths. These data suggest that dsRNA-mediated innate immune responses are length-dependent and longer molecules induce a more robust response. There were also some differences in the cells response to in vitro transcribed dsRNA compared to poly I:C. This provides important information for potential dsRNA-based antiviral therapies and vaccine adjuvants. © 2015 Elsevier Ltd. All rights reserved.

Keywords: dsRNA Innate immunity Rainbow trout Antiviral Interferon ISG

1. Introduction Almost all viruses produce double-stranded (ds)RNA at some point during their replication cycle [16]. DsRNA is a potent signaling molecule; a single dsRNA molecule is sufficient to induce an innate immune response [27]. Healthy vertebrate cells do not produce endogenous dsRNA greater than ~30 bp in length while viruses produce longer molecules; this difference allows cells to discriminate self-vs. viral-dsRNA molecules [9]. Polyinosinic:polycytidylic acid (poly I:C) is a viral dsRNA mimic and toll-like receptor 3 (TLR3) agonist that is regularly used as a stimulant in fish to study innate antiviral responses, both in vitro and in vivo [26,10,7,4]. Although poly I:C is a potent immunostimulant, it lacks the natural structures, base composition, and sequence variations of viral dsRNA [26]. Because of these differences poly I:C does not accurately reflect the functional properties of natural dsRNA fragments [26]. Importantly, the innate immune responses induced by in vitro transcribed dsRNA differ compared to poly I:C. Though not well

* Corresponding author. E-mail address: [email protected] (S.J. DeWitte-Orr). http://dx.doi.org/10.1016/j.fsi.2015.07.012 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

characterized, phospho-interferon regulatory factor 3 (IRF3) expression kinetics differed between poly I:C and in vitro transcribed dsRNA molecules based on a viral genome [19]. Gene expression levels were also found to differ in murine embryonic fibroblasts treated with either poly I:C or viral in vitro transcribed dsRNA [11]. The differences in the innate immune response between poly I:C and in vitro transcribed dsRNA molecules has yet to be elucidated in fish cells. Not only do innate immune responses differ between poly I:C and viral dsRNA, it also is difficult to determine lengths effects using poly I:C. The length of a dsRNA molecule is an important factor in modulating the innate immune response. Studies in mammals have shown that longer dsRNA molecules (>1000 bp) are able to induce a stronger innate immune response compared with shorter molecules (<1000 bp) [8,11]. While high and low molecular weight poly I:Cs are available commercially, all poly I:C preparations contain a mixture of nucleic acid lengths that run as a smear on an agarose gel, making it challenging to determine length effects using this molecule. Thus in vitro transcribed molecules are superior for determining length effects as they can be synthesized at clearly defined lengths. Little is known of dsRNA length effects in

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fish. TLR22, a fish surface receptor for dsRNA, demonstrated length effects in induction of IFN-b when expressed in human cells, suggesting that length may play a role in the magnitude of immune responses in fish cells expressing fish receptors for dsRNA [28]. DsRNA sensed by the cell can induce type I interferons (IFNs) [27]. Rainbow trout (Oncorhynchus mykiss) have at least 5 subgroups of IFNs (IFN a-f), which can be classified into two groups (group I and II) based on the number of cysteines present within the mature peptide (2 and 4 respectively). IFN1 and IFN2 used in the present study are members of group I IFNs [34,3,44]. IFNs signal in an autocrine or paracrine manner through their cognate receptor to stimulate the expression of interferon-stimulated genes (ISGs). ISGs function to limit virus replication within the cell. Many ISGs have been identified in teleost fish, including virus-induced gene (vig) 1e10 and Mx1-3 [31]. In DNA vaccinated rainbow trout there was a correlation between protection and Mx expression [29]. Mxs have been shown to limit virus replication in Atlantic salmon and Japanese flounder [23,2]. The vigs are a group of proteins that are upregulated during virus infections. Vig3 has been identified in rainbow trout and is an ubiquitin-like protein that has high similarity to the mammalian ISG15 [31,42]. ISG15 has demonstrated antiviral activity against a variety of viruses in mammals and target proteins through an ISGylation pathway, a protein modification process similar to ubiquitination [42,45]. Vig4 has also been identified in rainbow trout and is similar to the mammalian ISG56/ ISG58 [31]. The mammalian protein homologue ISG56 has been shown to interfere with protein synthesis [36,42]. The accumulation of ISGs within a cell results in the establishment of an antiviral state. The antiviral state inhibits the replication of DNA and RNA viruses [37]. This has been observed in both in vitro and in vivo studies in mammals and fish [18,37,41]. Poly I:C can induce an antiviral state in both fish and fish cell lines. For example, poly I:C transfected into a Chinook salmon embryo cell line resulted in an antiviral state against infectious pancreatic necrosis virus [18]. Four species of whole salmon were injected with poly I:C and demonstrated antiviral responses when challenged with infectious hematopoietic necrosis virus or erythrocytic necrosis virus [13]. While poly I:C-induced innate immune responses are relatively well characterized in fish, nothing is known of fish cell's ability to detect and respond to dsRNA containing a fish virus genome sequence nor is anything understood about the influence of dsRNA length on the IFN-mediated immune responses in fish. The present study aimed to measure if in vitro transcribed dsRNA molecules synthesized using the viral hemorrhagic septicemia virus (VHSV)IVb genome, firstly induced an IFN-mediated innate immune response in rainbow trout cells and secondly induced this response in a length dependent manner. DsRNA-induced innate immune effects were tested in RTG-2, a rainbow trout gonadal cell line that has previously been shown to respond to poly I:C [40]. Poly I:C, including high and low molecular weight mixtures, was used as a positive control. The present study demonstrates that both poly I:C and in vitro transcribed VHSV dsRNA were both able to robustly induce an innate immune response and antiviral state. While both types of dsRNA were potent inducers, there were notable differences between the synthetic dsRNA and sequence specific in vitro

N (1215)

P (669)

M (606)

transcribed dsRNA. Length effects were observed between low and high molecular weight poly I:C and with different lengths of in vitro transcribed dsRNA. 2. Materials and methods 2.1. Cell lines The two cell lines used in this study were the rainbow trout gonad (RTG-2), and the transgenic RTG-2 (RTG-P1). RTG-2 was obtained from N. Bols (University of Waterloo) and RTG-P1 was obtained from B. Collet (University of St. Andrews). All cell lines were grown in 75 cm2 plastic tissue culture flasks (BD Falcon, Bedford, MA) at room temperature in Leibovitz's L-15 media (HyClone, Logan, UT) supplemented with 10% v/v fetal bovine serum (FBS) (Fisher Scientific, Fair Lawn, NJ) and 1% v/v penicillin/ streptomycin (P/S) (10 mg/mL streptomycin and 10000U/mL penicillin) (Fisher Scientific). RTG-P1 media was supplemented with an additional 100 mg/mL of G418 neomycin (SigmaeAldrich, St Louis, MO). 2.2. Virus propagation Viral hemorrhagic septicemia virus (VHSV)-IVb (strain U13653) was propagated on monolayers of epithelioma papulosum cyprinid (EPC) cells. Virus containing media (L-15 with 5% v/v FBS (Fisher Scientific)) was collected 4e7 days post-infection, filtered through a 0.45 mm filter (Nalgene, Rochester NY, USA) and kept frozen at
20  C for short-term storage and
80  C for long-term storage. The 50% tissue culture infective dose (TCID50)/mL values were estimated according to the method of Reed and Muench [35]. 2.3. Poly I:C Poly I:C was stored in stock solutions of 10 mg/mL for regular (SigmaeAldrich, St Louis, MO, USA) and low molecular weight (LMW) poly I:C (InvivoGen, San Diego, CA, USA) and 1 mg/mL for high molecular weight (HMW) poly I:C (InvivoGen) diluted in phosphate buffered saline (PBS) (HyClone) based on manufacturers instructions. Average length was estimated based on the brightest size of the smear observed on a 1% agarose (Fisher Scientific, Fair Lawn, NJ, USA) gel compared to 5 ml of O'GeneRuler 1 kb PLUS DNA Ladder (Fermentas, Carlsbad, CA, USA). The gel was stained with 0.5 mg/mL ethidium bromide (Fisher Scientific). Molarity was calculated using the average length for each poly I:C molecule, as previously described [11], and is therefore an approximate value. Poly I:C aliquots were stored at
20  C. 2.4. In vitro transcribed dsRNA DsRNA was synthesized using a section of the VHSV genome as a template (Fig. 1). The dsRNA molecules were 240 bp (v200) and 1264 bp (v1200) in length, and synthesized from the G gene (v200) and the N and G genes (v1200) respectively. The dsRNA molecules were synthesized in vitro using the MegaScript RNAi kit (Ambion by

G (1524)

v1200

NV (369)

L (5955)

v200

Fig. 1. Section of VHSV genome amplified as template for in vitro transcribed dsRNA. A representative figure of the viral hemorrhagic septicemia virus (VHSV) genome in the 30 50 orientation, the length of each gene (bp) is given in brackets. The section amplified for use in synthesis of the v1200 and v200 dsRNA molecules is indicated. Genome sequence accession #: GQ385941.

S.J. Poynter, S.J. DeWitte-Orr / Fish & Shellfish Immunology 46 (2015) 557e565

Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Briefly, EPC cells were infected with 3.16  104 TCID/ mL of VHSV for 4 days. Total RNA was extracted using TRIzol reagent following manufacturers instructions (Life Technologies). cDNA was synthesized using GoScript reverse transcriptase (Promega, Madison, WI) following manufactures instructions; the reaction was primed using random hexamers (SigmaeAldrich). Fragments of the VHSV-IVb genome were amplified by RT-PCR using sequence specific primers linked 50 to a T7 RNA polymerase promoter (3′-TAATACGACTCACTATAGGG-5′), Table 1. PCR reactions contained: 2 ml of cDNA, 1 Green GOTaq Flexi Buffer (Promega), 0.2 mM each dNTP (SigmaeAldrich), 1.25U GoTaq Flexi DNA Polymerase (Promega), 1.5 mM MgCl2 (Promega), 0.5 mM forward primer (Sigma Aldrich), 0.5 mM reverse primer (Sigma Aldrich) and up to 25 ml nuclease-free water (Fisher Scientific). The PCR reactions were run using a T100 Thermal Cycler (Bio-Rad). T7 RNA polymerase synthesized complimentary RNA strands from the template that were annealed to form dsRNA. The dsRNA reaction was treated with RNase and DNase I to remove any template DNA or ssRNA and then filter purified. To quantify the dsRNA molecules, 1/400th of the reaction was visualized on a 1% agarose (Fisher Scientific) gel compared to 5 ml of O'GeneRuler 1 kb PLUS DNA Ladder (Fermentas). The gel was stained with 0.5 mg/mL ethidium bromide (Fisher Scientific) and dsRNA was quantified through gel densitometry (ImageLab software, Bio-Rad) and verified by spectrophotometry with the NanoDrop Lite (Thermo Scientific). 2.5. qRT-PCR 2.5.1. RNA extraction and cDNA synthesis RTG-2 cells were seeded at 5  105 cells/well in a 6-well plate and allowed to attach over night. Cells were treated with 1.5 nM poly I:C (HMW or LMW) or in vitro transcribed dsRNA (v200 or v1200) in growth media for 3 h or 6 h. 1.5 nM was used as this molarity was found to effectively stimulate the Mx-promoter in the reporter assay (described below). 3 h, 6 h, and 24 h were initially surveyed, however as there were no differences seen between LMW and HMW poly I:C at 24 h this time point was not pursued. RNA was extracted using the GenElute mammalian total RNA miniprep kit (SigmaeAldrich) following manufacturer's instructions. RNA was treated with an on-column DNase I digestion

Table 1 Primer sequences, including product size, annealing temperature (TA), and reference for primers or accession number. Gene or Primer sequence 5e30 fragment

b-Actin IFN1 IFN2 Mx1 Mx3 Vig3 Vig4 v1200 v200

Product TA Reference or length ( C) Accession number

F-GTCACCAACTGGGACGACAT 174 R-GTACATGGCAGGGGTGTTGA F-AAAACTGTTTGATGGGAATATGAAA 141 R-CGTTTCAGTCTCCTCTCAGGTT F-AGTTCCTGTGTATCACCTGTCG 182 R-GATGCTCAGTACATCTGTCCCA F-CGGAGTTCGTCTCAACGTCT 140 R-CCCTTCCACGGTACGTCTTC F-TGAGGCCATTAAGCAGGTGA 151 R-TGGTAAGGGTCGGTCGTCT F-ATGGAAAGGCAGAGGCTGTC 128 R-TGAGTGGGTTCTGTAATCAGCA F-GGGCTATGCCATTGTCCTGT 151 R-AAGCTTCAGGGCTAGGAGGA F-ACGGACAAGCGAAGGACTAC 1264 R-TCGCATGATCTGGCCATCAA F-TTCAGATGAGGGGAGCCACA 240 R-TCGCATGATCTGGCCATCAA

55

NM_001124235

55

[5]

55

[5]

55

NM_001171901.1

55

U47946.1

55

NM_001124332.1

55

NM_001124333.1

62

GQ385941

52

GQ385941

559

set (SigmaeAldrich). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). 1 mg of RNA, 1 reverseetranscriptase reaction mix, and up to 20 ml DNA quality water were included in each reaction. cDNA was diluted 1 in 10 in DNA quality water prior to usage in PCR reactions. 2.5.2. qPCR reactions All PCR reactions contained: 2 ml of diluted cDNA, 2 SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA), 0.2 mM forward primer (Sigma Aldrich), 0.2 mM reverse primer (SigmaeAldrich) and nuclease-free water to a total volume of 10 ml (Fisher Scientific). The primer sequence, accession number, and expected product size for each primer set are listed in Table 1 qPCR reactions were performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad). The program used for all qPCR reactions was: 98  C 2 min, 40 cycles of 98  C 5 s, 55  C 10 s, followed by 95  C for 10 s. A melting curve was completed from 65  C to 95  C with a read every 5 s. Product specificity was determined through single PCR melting peaks. Data were analyzed using the DDCt method. Specifically, gene expression was normalized to the housekeeping gene (b-actin) and expressed as fold change over the control group, control expression was set to 1. 2.6. Mx1-promoter reporter assay The Mx1-promoter reporter assay was modified from Collet and colleagues [6]. RTG-P1 cells were seeded at a density of 5  105 cells/well in a 6-well plate (BD Falcon). Cells were allowed to attach overnight. Media was removed from all wells, cells were treated with growth media containing 1.5 nM of either regular poly I:C (SigmaeAldrich), HMW poly I:C (InvivoGen), LMW poly I:C (InvivoGen), v1200, v200 in vitro transcribed molecules or untreated control media. After 24 h, cells were dissociated and collected through centrifugation at 1400  g for 5 min. The cell pellet was re-suspended in 100 ml of Steady-Glo luciferase substrate (Promega) and incubated for 40 min in the dark. 90 ml of the substrate/cell mixture was plated into a 96-well plate (BD Falcon) and luminescence was measured in relative luminescent units (RLUs) using a Synergy HT plate reader (BioTek, Winooski, VT). Time and molar concentration were optimized in preliminary experiments; 24 h was found necessary to accumulate significant measurable luciferase and 1.5 nM the minimum amount of dsRNA that was found to strongly activate the Mx1 promoter. 2.7. Antiviral assay RTG-2 cells were seeded at 2  103 cells/well in 96-well plates (BD Falcon). Cells were allowed to attach overnight. 1.5 nM dsRNA was found to induce 100% protection in the antiviral assay, thus 1 in 5 serial dilutions of dsRNA were performed beginning at 1 nM. Media was removed from wells and replaced with growth media containing v1200, v200, LMW poly I:C, HMW poly I:C or control media. After 6 h the media was removed and the cells were rinsed with PBS (HyClone). Cells were infected with VHSV at a multiplicity of infection (MOI) of 8 (an MOI that without dsRNA-induced protection caused significant CPE compared with uninfected controls, p < 0.05), in 5%v/v FBS (Corning) media. Untreated control cells were both infected and uninfected. After a 5-day incubation at 17  C, cells were rinsed with PBS (HyClone) and cell viability was measured using 5-carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM, Invitrogen), as previously described. CFDA-AM is a cell viability indicator that measures cell membrane integrity [15]. Images of the cells following completion of the antiviral assay were taken using a Nikon Eclipse TS100 at 100 magnification.

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(Fig. 2B). DsRNA runs slightly slower than DNA on an agarose gel, thus size comparisons to the DNA ladder are approximate [9].

2.8. dsRNA uptake assay In vitro transcribed dsRNA was labeled with Alexafluor 488 using the Ulysis nucleic acid labeling kit (Invitrogen, Carlsbad, CA). Excess labeling reagent was removed using Micro Biospin P-30 columns (BioRad). DsRNA was diluted in PBS to a final concentration of 1 mg/mL 3  104 RTG-2 cells were seeded in 6-wells of a 96well plate (BD Falcon). Cells were treated with 1 mg/mL of dsRNA in PBS or PBS alone for control. After a 1 h incubation dsRNA containing PBS was removed and PBS was added to 1/2 the wells while 0.025% trypan blue was added to the other 1/2 (SigmaeAldrich) to quench external fluorescence. Total fluorescence (PBS) and internal fluorescence (trypan blue) were measured using a Synergy HT plate reader (BioTek, Winooski, VT, USA). 2.9. Statistical analysis All data presented is derived from three independent experiments. Data were statistically analyzed using a one-way ANOVA and Tukey's post-hoc test when multiple samples were compared, or a Student's t-test performed when two samples were compared using Kaleidagraph software (Synergy Software, Reading, PA, v4.1.0). qRT-PCR data was log2 transformed prior to analysis. 3. Results 3.1. DsRNA molecules used in the study To confirm dsRNA lengths, 50 ng of each in vitro transcribed molecule and 1 mg of each type of poly I:C was run on an agarose gel and stained with ethidium bromide; 10 mg of regular poly I:C was run on a gel to ensure 1 mg was sufficient for estimating average length (Fig. 2A). All poly I:C produced a smear of mixed length nucleic acids. LMW poly I:C was estimated to have an average length of 300 bp, HMW poly I:C to have an average length of 3000 bp, and regular poly I:C to have an average length of 500 bp. In vitro transcribed dsRNA was synthesized from a cDNA template amplified from the VHSV RNA genome. Resulting dsRNA fragments were 240 bp and 1264 bp long and referred to as v200 and v1200 respectively. The dsRNA products were confirmed to be single bands of the appropriate length by agarose gel electrophoresis

500bp 300bp

v200

1μg

10μg

HMW

LMW 3000bp

Reg.

v1200

B In vitro

A Poly I:C

1500bp

200bp

Fig. 2. dsRNA synthesis and characterization. A: 1 mg of high molecular weight (HMW), low molecular weight (LMW), or regular (reg.) poly I:C were run on a 1% agarose gel to allow for estimation of average lengths; 10 mg of reg. poly I:C was also included. B: In vitro transcribed dsRNA, v200 and v1200, was synthesized using the VHSV genome as a template. 50 ng of the final product was run on a 1% agarose gel. O'GeneRuler 1 kb PLUS DNA Ladder (Thermo Scientific) was run on both gels that were stained with ethidium bromide.

3.2. dsRNA induces the production of IFN and ISG transcripts in a length-dependent manner The ability for poly I:C (LMW, HMW) and in vitro transcribed dsRNA (v200, v1200) to induce IFNs and ISGs was measured using qRT-PCR. RTG-2 cells were treated with equal molar amounts of dsRNA (1.5 nM) for 3 h or 6 h and transcript levels were measured (Figs. 3 and 4). Equal molar amounts were chosen to ensure the same number of molecules were added to the cells in order to determine effects of length on IFN and ISG induction. Two IFNs, IFN1 and IFN2, were measured as well as four ISGs: Mx1, Mx3, vig3 and vig4. At 3 h HMW and LMW dsRNA induced expression of all genes, with the exception of Mx1 (Fig. 3A). Significant difference in expression between lengths was observed for IFN2, Vig3, and Vig4. While not significant the trend was still visible for IFN1, Mx1 and Mx3. At 6 h there was a decline in the overall expression of IFN1 and IFN2, and the length effect was now significant for both IFN genes (Fig. 3B). All ISGs increased in expression compared to untreated controls by 6 h and there was significantly different expression between HMW and LMW poly I:C with HMW poly I:C being the stronger inducer for all ISGs tested. The pattern of IFN and ISG transcript induction was different with the in vitro transcribed dsRNA molecules. Similar to poly I:C, v200 and v1200 also induced IFN and ISG expression at the transcript level in RTG-2 cells (Fig. 4). At 3 h IFN1, IFN2, vig3 and vig4 transcript induction could be detected over untreated controls, and there was a significant difference in expression between v200 and v1200 treatments for these genes, with v1200 dsRNA inducing a stronger response. Mx1 and Mx3 induction was not detected at 3 h (Fig. 4A). At 3 h IFN1 and IFN2 transcripts were at higher levels in cells treated with v200 and v1200 compared with LMW and HMW poly I:C treated cells, particularly with v1200 which induced IFN1 and IFN2 transcript levels significantly higher than LMW and HMW poly I:C (p < 0.01). At 6 h, all IFN and ISG transcripts were induced over untreated controls; however, there were no significant differences in levels between the two lengths of dsRNA (v200 vs. v1200) (Fig. 4B). IFN1 and IFN2 expression decreased at 6 h compared to 3 h for both lengths while all ISGs showed an overall increase in expression at 6 h compared to 3 h.

3.3. dsRNA induces Mx1-promoter activation in a length-dependent manner Levels of ISG promoter activation were measured using the transgenic RTG-2 cell line RTG-P1. RTG-P1 cells express the firefly luciferase gene under the control of the Mx1-promoter. Cells were treated with equal molar amounts of dsRNA (1.5 nM) for 24 h and total luminescence was measured. All dsRNA treatments significantly induced the activation of the Mx1 promoter and subsequently the production of luciferase and measurable luminescence (Fig. 5). HMW poly I:C and v1200 in vitro transcribed dsRNA generated greater amounts of promoter activation as compared to their shorter counterparts, LMW poly I:C and v200 respectively. When regular poly I:C (average length 500 bp) was included as a treatment alongside in vitro transcribed dsRNA molecules it was found to produce activation levels between those of the v200 and v1200 molecules, however not significantly different from either (Fig. 5A). Neither was regular poly I:C significantly different from LMW poly IC (p ¼ 0.22). All dsRNA treatments up-regulated promoter activity compared to mock treated controls (p < 0.001).

S.J. Poynter, S.J. DeWitte-Orr / Fish & Shellfish Immunology 46 (2015) 557e565

B: 6h

A: 3h IFN1

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***

100 0 HMW

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4000

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HMW

800

5000

**

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0 LMW

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Mx3

5

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Relative normalized expression

0

*

60

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50

Relative normalized expression

80

100

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*

150

150

200

IFN2

IFN1

*

200

250

300

561

0 LMW

HMW

0 LMW

HMW

LMW

HMW

Fig. 3. Extracellular poly I:C results in the length-dependent induction of IFN and ISGs. RTG-2 cells were treated with 1.5 nM high molecular weight (HMW) or low molecular weight (LMW) poly I:C for A: 3 h or B: 6 h. Interferon (IFN) or IFN-stimulated gene (ISG) or transcript levels were measured using real time PCR and reported as relative normalized expression, normalized to the housekeeping gene b-actin and mock treated controls. These data are the average of three independent experiments ± SEM. Statistical analysis was performed on log2-transformed values using a one-way ANOVA with a Tukey's post-hoc test, alpha 0.05. *p < 0.05, **p < 0.01, p < 0.001.

3.4. dsRNA induces an antiviral state in a length-dependent manner An antiviral assay was used to measure the ability of dsRNA molecules with different lengths to induce an antiviral state. RTG2 cells were pretreated with dsRNA concentrations from 1.0 nMe0.0016 nM and then infected with VHSV for 6 days. DsRNA, both synthetic and in vitro transcribed, induced a robust antiviral state. This can be seen both through increased cell viability, measured by CFDA-AM (Fig. 6) and through reduced cytopathic effects. From 1.0 nM to 0.04 nM there were no significant differences compared to uninfected controls. Additionally at these concentrations there was no significant differences between lengths of molecules, although there does appear to be a length-dependent trend. At the two lowest concentrations tested, 0.008 nM and 0.0016 nM, there was significantly more protection seen when cells were treated with the longer dsRNA molecules (HMW poly I:C and v1200) compared with their shorter counterparts (LMW poly I:C and v200). 3.5. DsRNA entry is not length dependent The quantity of extracellular dsRNA capable of entering the cell was measured to assess whether the dsRNA length effects observed were due to differences in ability to enter the cell. RTG-2 cells were treated with in vitro transcribed dsRNA molecules labeled with Alexa Fluor 488. After a 1 h treatment dsRNA was removed and external fluorescence was quenched using trypan blue [12] or left unquenched to obtain intracellular and total fluorescence values respectively. Fluorescence was measured using a fluorescence plate

reader and data are presented as intracellular fluorescence normalized to total fluorescence. Approximately 35% of total fluorescence was found to be intracellular for both dsRNA molecules, with no significant difference in entry between the two dsRNA lengths, suggesting that both v200 and v1200 enter the cells in equal amounts (Fig. 7). 4. Discussion RTG-2 cells respond robustly to treatment with extracellular dsRNA. This response was seen when cells were treated with the synthetic viral dsRNA mimic poly I:C and with the VHSV sequence specific in vitro transcribed dsRNA. Previous data has shown that RTG-2 cells are capable of mounting an antiviral response when treated with poly I:C [40]. To the author's knowledge this is the first time low (LMW) and high molecular weight (HMW) poly I:C have been used in fish cells to explore innate immunity and length effects. To increase biological relevance, dsRNA molecules were synthesized in vitro using a viral genome sequence template and making molecules of a defined length. Two different lengths of dsRNA were produced; 1264 bp (v1200) and 240 bp (v200), with a VHSV based sequence (Fig. 1). The lengths were chosen as representative long and short dsRNA molecules respectively. While the v200 was considered the ‘short’ molecule in this study compared to the v1200, when compared to endogenous dsRNA (<30 bp) v200 is still a long dsRNA molecule that is capable of being sensed by host cell pattern recognition receptors (PRRs) [11,9]. To avoid the number of molecules acting as a confounding variable, cells were treated with equal nanomolar concentrations as opposed to weight

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A: 3h

B: 6h IFN1

IFN2

*

5000 4000

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v1200

Mx1

100 v200

0 v200

Relative normalized expression

v200

IFN2 80

60

1500

2000

Relative normalized expression

IFN1 100

v1200

v200

v1200

Fig. 4. Extracellular in vitro transcribed dsRNA results in the length-dependent induction of IFN and ISGs. RTG-2 cells were treated with 1.5 nM in vitro transcribed dsRNA, v200 or v1200 lengths for A: 3 h or B: 6 h. Interferon (IFN) or IFN-stimulated gene (ISG) transcript levels were measured using real time RT-PCR and reported as relative normalized expression, normalized to the housekeeping gene b-actin and mock treated controls. These data are the average of three independent experiments ± SEM. Statistical analysis was performed on log2-transformed values using a one-way ANOVA with a Tukey's post-hoc test, alpha 0.05. *p < 0.05, **p < 0.01.

A

B 120

120

***

** 100 %HMW RLUs

% v1200 RLUs

100 80 60

80 60

40

40

20

20 0

0 Control

v200

Poly I:C

v1200

Control

LMW

HMW

Fig. 5. dsRNA induced Mx1-promoter activation in RTG-P1 cells. RTG-P1 cells containing a luciferase gene under the control of the Mx1 promoter were stimulated with 1.5 nM dsRNA, A v200 dsRNA or v1200 dsRNA, based on the VHSV genomic sequence B LMW, low molecular weight poly I:C; HMW, high molecular weight poly I:C;. After 24 h a luciferase substrate was added to samples and relative luminescent units (RLUs) were measured. Data is presented as % of the strongest activator. Data represents three independent trials ± SEM and was analyzed using a one way ANOVA and Tukey's post hoc test, alpha 0.05. All dsRNA treatments up-regulated promoter activity compared to mock treated controls (p < 0.001). *p  0.05, **p  0.01, ***p  0.001.

concentrations. This ensured an equal number of molecules/cell for each dsRNA length; thus differences in responses can be attributed to length as opposed to differences in number of molecules/cell. Previous studies in mammals have used molar concentrations of in vitro transcribed dsRNA molecules when exploring dsRNA length effects [12,1].

The present study provided several different lines of evidence supporting the hypothesis that both poly I:C and in vitro transcribed dsRNA are able to induce IFN-mediated innate immune responses in a length dependent manner in rainbow trout cells. Following dsRNA sensing, cells first make IFNs, which in turn trigger the production of ISGs. Three type 1 IFNs (IFN1, IFN2, IFN3/4) were

S.J. Poynter, S.J. DeWitte-Orr / Fish & Shellfish Immunology 46 (2015) 557e565

Fold change from infected control

A

Fold change from infected control

B

3 2.5

*

2

*

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1

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0.5 0 Uninf.

1.0

0.20 0.04 [dsRNA (nM)]

0.008

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2.5

*

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v1200

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0.5 0 Uninf.

1.0

0.20 0.04 [dsRNA (nM)]

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Fig. 6. dsRNA induces a length-dependent antiviral state in RTG-2. RTG-2 cells were pretreated with molar amounts of A high molecular weight (HMW) or low molecular weight (LMW) poly I:C or B in vitro transcribed dsRNA of 240 bp (v200) or 1264 bp (v1200) in length. The dsRNA was removed after 6 h and cells were infected with VHSV (MOI: 8). 6 days post-infection cell viability was measured using CFDA-AM, a fluorescent indicator of cell membrane integrity. Data include three independent trials and are presented as fold change over an untreated, infected control ± SEM; an untreated, uninfected control was included to demonstrate a healthy cell monolayer. Different lengths of dsRNA were compared using a student t-test, *p < 0.05, **p < 0.01; groups within a treatment were compared with a one way ANOVA and Tukey's post-hoc test, alpha 0.05, A ¼ significantly different from control, AP < 0.01, AAp < 0.001.

Intracellular fluorescence as % Total fluorescence

45

p = 0.25

40 35 30 25 20 15 10 5 0 v200

v1200

Fig. 7. Cellular uptake of in vitro transcribed dsRNA. In vitro transcribed dsRNA was labeled with Alexa-Fluor 488. Cells were incubated with dsRNA, either 1264 bp (v1200) or 240 bp (v200) in length, and extracellular fluorescence was quenched with trypan Blue. Data is presented as the percentage of intracellular fluorescence out of total fluorescence. Data represents three independent experiments ± SEM. No significant difference as determined by a Student's t-test.

initially investigated; however only IFN1 and IFN2 were induced at the transcript level in dsRNA-treated RTG-2 cells (data not shown). This was expected as IFN3/4 is expressed in rainbow trout ovaries but has not been detected in RTG-2 [43]. Previous studies have found both IFN1 and IFN2 transcripts were strongly upregulated in

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RTG-2 cells in response to regular poly I:C [43]. Mx1 and Mx3 are two ISGs that have previously been shown to be upregulated in RTG-2 cells in response to regular poly I:C [10,40]. Vig3 and vig4 were included in the ISG panel because they have important human homologues (ISG15 and ISG56 respectively) and have been identified in virus-infected rainbow trout leukocytes from head-kidney [31]; however, this is the first time they have been measured in response to any dsRNA treatment in fish cells. Thus IFN and ISG induction by LMW and HMW poly I:C as well as in vitro transcribed dsRNA occurs in rainbow trout cells. Both IFN1 and IFN2 transcripts were upregulated in RTG-2 in response to poly I:C, and in vitro transcribed dsRNA (Figs. 3 and 4). Interestingly at 3 h the v1200 molecule was the strongest inducer of type 1 IFN, even though HMW poly I:C is a longer dsRNA molecule. This finding suggests that length is not the only factor contributing to the intensity of the IFN response to dsRNA, and sequence may also play a role. By 6 h all dsRNA treatments had a lower IFN expression than at 3 h, which could correspond to the cyclic nature of IFN transcript expression [30]. All ISGs were more strongly expressed by 6 h compared to 3 h (Figs. 3 and 4). As the cell needs time to produce IFN and respond to the cytokine it is not surprising that the ISGs took longer to be induced. At 3 h there was higher expression of vig3, vig4, IFN1, and IFN2 when cells were stimulated with the v1200 vs. the v200 molecule, and higher expression of vig3, vig4, and IFN2 when cells were treated with HMW vs. LMW poly I:C. By 6 h HMW and LMW length effects are present for all immune genes but no length effects are seen for the in vitro molecules. This suggests that in vitro transcribed dsRNA triggers an IFN response quickly and more robustly compared with poly I:C; however, this stimulation is short lived and drops by 6 h. Poly I:C molecules are slower to induce an IFN response, but their effects on IFN and ISG transcript levels are longer lasting. Further research is required to understand the kinetic differences between these two dsRNA molecules. Next, ISG promoter activity was measured using the Mx1promoter reporter assay in RTG-P1 cells. Poly I:C has previously been shown to induce Mx1 promoter activation in RTG-P1 after 24 h at a concentration of 100 mg/ml [6]. The present study used the RTG-P1 in a similar manner; however in the present study cells were treated with equal molar amounts of poly I:C, regular (poly I:C), HMW and LMW, as well as in vitro transcribed dsRNA, v200 and v1200. All extracellular dsRNA was able to significantly activate the Mx1-promoter (Fig. 5). Consistent with what was seen in the qRT-PCR, longer dsRNA molecules stimulated the promoter more strongly compared with shorter lengths. These length effects observed at the promoter and transcript levels translated to length effects in the accumulated antiviral response mounted by the cells. Regular poly I:C has previously been used to stimulate an antiviral state within fish cells [18,17]. A main difference between previous assays and the assay used in the present study was the concentration of poly I:C used. Previous studies have used poly I:C concentrations such as 10 mg/mL and 50 mg/mL [17,10] to induce type I IFN responses in fish cells, whereas the present study has found that when infected with a moderate MOI (MOI ¼ 8) of VHSV these concentrations are unnecessary for establishing a complete antiviral state, and low nanomolar concentrations of dsRNA, between 0.132 mg/mL and 2.97 mg/mL are sufficient. HMW and LMW poly I:C and in vitro transcribed dsRNA molecules have not previously been shown to induce an antiviral state in fish cells. It was found that after a 6 h pretreatment with 1 nMe1.6pM dsRNA there was protection from VHSV infection suggesting an antiviral state was mounted in response to dsRNA (Fig. 6). Even at these low concentrations dsRNA there was still a robust antiviral state established; this reinforces how potent dsRNA is as an immunomodulating molecule, where in early studies it was

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found that a single dsRNA molecule induced IFN expression [27]. At the higher concentrations levels of protection between long and short dsRNA molecules were similar. At lower concentrations the longer dsRNA molecules (HMW poly I:C and v1200 dsRNA) induced a significantly more protective state compared to their shorter dsRNA counterparts (LMW poly I:C and v200) (Fig. 6). In order to induce a cellular response, extracellular dsRNA must enter the cell. It was hypothesized that perhaps longer dsRNA molecules could reach the cell and enter more efficiently than smaller dsRNA molecules resulting in a stronger response. However, when similar numbers of v200 and v1200 were added to RTG2 cells there was no difference observed in entry (Fig. 7). It can be thus concluded that the length effects observed must be the result of difference in intracellular sensor activation or subsequent signaling pathway activation. Further studies are underway to determine differences in downstream pathways that could account for the length effects observed; however, several hypotheses to explain dsRNA length effects are described below. From these results two conclusions can be made. Firstly, dsRNAinduced, IFN-mediated innate immune responses are dsRNA length dependent in fish cells; specifically longer dsRNA molecules induce a stronger response. Secondly, when comparing dsRNA molecules of similar lengths, there are differences in responses between poly I:C and in vitro transcribed dsRNA molecules. These results are consistent with what has previously been observed in mammalian cells treated with poly I:C and in vitro transcribed dsRNA. DsRNA length effects were observed in vesicular stomatitis virus-infected mouse embryonic fibroblasts (MEFs), where longer in vitro transcribed dsRNA molecules resulted in lower EC50 values than did molecules of shorter lengths and in uninfected MEFs IFN and ISG transcripts were induced in a dsRNA length dependent manner [11]. A study in rabbits found that longer dsRNA molecules induced greater somnogenic and pyrogenic responses compared to shorter molecules [14]. Similar to what has been observed here in fish, poly I:C and in vitro transcribed dsRNA induce similar but not exactly the same innate immune responses in mammals [11,19]. A study in murine cells examined the expression of a panel of ISGs in response to poly I:C and similar length in vitro molecules [11]. It was found that poly I:C induced some of the ISGs within the panel more strongly than in vitro molecules, however some ISGs were induced more strongly in response to in vitro transcribed molecules [11]. Another study in murine cells looked specifically at IFN-b (a type I IFN) production and found greater production in response to in vitro molecules of similar length compared to poly I:C [20]. In RTG-2 cells the present study found that v1200 induced more type I IFN transcript (IFN1 and IFN2) compared with HMW poly I:C, suggesting the pathway in fish may be similar to that in mammals. One possible hypothesis for the observed difference between poly I:C and in vitro transcribed dsRNA may be at the dsRNA-sensor level. There is evidence that receptor clustering, which can occur to a greater extent with longer dsRNA molecules, can trigger a stronger IFN response. This was shown with TLR3 in mammals [24]. In mammalian TLR3 the shortest length for activation was 40e50 bp but longer molecules, >100 bp, resulted in greater binding and activation [24]. It is also possible that different lengths of dsRNA could activate different dsRNA sensors. While very little is understood of length specificities for dsRNA sensors in any animal, fish in particular are not well studied. One study looking at Fugu (Takifugu rubripes) TLR3 and TLR22 expression in HEK293 cells found that FuTLR3 responded most strongly to a 200 bp in vitro transcribed dsRNA while FuTLR22 responded most strongly to a 1000 bp molecule [28]. In mammals RIG-I recognizes shorter dsRNA molecules (<1000 bp) and MDA5 recognizes longer molecules (>1000 bp) [21,32]. RIG-I has not been cloned in rainbow trout, however MDA5 has been identified in RTG-2 cells and was

upregulated in response to poly I:C stimulation [4]. Clearly, more research into fish PRRs will be needed to confirm if there are similar length effects seen in fish as in mammals. DsRNA sensors may also have different affinities for poly I:C vs. in vitro dsRNA that would be length independent. Fugu TLR3 when transfected into human cells was found to induce more IFN promoter activity when bound to in vitro transcribed dsRNA compared with poly I:C. Human TLR3 induced stronger IFN promoter activity in response to poly I:C vs. in vitro transcribed dsRNA. The template for the dsRNA in this study was measles virus [28]. It may be that fish PRRs are more sensitive to in vitro transcribed dsRNA compared to poly I:C, while the opposite may be the case in humans. The end result of these early differences in IFN and ISG production between poly I:C and in vitro transcribed dsRNA did not result in differences once an antiviral state was formed. The present study shows that both types of dsRNA molecules are able to induce an IFN-mediated antiviral response that is length dependent, and while there are some temporal differences in gene expression, there were no dramatic differences observed between poly I:C and in vitro transcribed molecules in mounting an antiviral state. The results from this study contribute to a better understanding of the host response to dsRNA. In particular, this is the first study to demonstrate that HMW and LMW poly I:C as well as in vitro transcribed dsRNA are able to induce IFNs, ISGs and an antiviral state in rainbow trout cells. It is also the first time that a dsRNA length effect has been observed in fish. These findings highlight the efficacy of using in vitro transcribed dsRNA as an IFN-inducer in fish cells, which has implications for using dsRNA molecules as immunomodulators in fish. Poly I:C has been used as a possible antiviral therapy in fish [39]. The present study would suggest that in vitro transcribed dsRNA molecules could also be used in this application, and perhaps with better results, as length and sequence can be precisely determined. Further work investigating the effects of in vitro transcribed dsRNA in vivo is currently underway. References [1] M. Binder, F. Eberle, S. Seitz, N. Mucke, C.M. Huber, N. Kiani, L. Kaderali, V. Lohmann, A. Dalpke, R. Bartenschlager, Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acidinducible gene-I (RIG-I), J. Biol. Chem. 286 (2011) 27278e27287. [2] C.M.A. Caipang, I. Hirono, T. Aoki, In vitro inhibition of fish rhabdoviruses by Japanese flounder Mx, Virology 317 (2) (2003) 373e382. [3] M. Chang, P. Nie, B. Collet, C.J. Secombes, J. Zou, Identification of an additional two-cysteine containing interferon in rainbow trout Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts, Immunogenetics 61 (2009) 315e325. [4] M. Chang, B. Collet, P. Nie, K. Lester, S. Campbell, C.J. Secombes, J. Zou, Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in rainbow trout (Oncorhynchus mykiss), J. Virol. 85 (16) (2011) 8403e8412. [5] E. Chaves-Pozo, J. Zou, C.J. Secombes, A. Cuesta, C. Tafalla, The rainbow trout (Oncorhynchus mykiss) interferon response in the ovary, Mol. Immunol. 47 (9) (2010) 1757e1764. [6] B. Collet, P. Boudinot, A. Benmansour, C.J. Secombes, An Mx1 promoter- reporter system to study interferon pathways in rainbow trout, Dev. Comp. Immunol. 28 (8) (2004) 793e801. [7] J. Congleton, B. Sun, Interferon-like activity produced by anterior kidney leucocytes of rainbow trout stimulated in vitro by infectious hematopoietic necrosis virus or Poly I: C, Dis. Aquat. Org. 25 (1996) 185e195. [8] O. De Bouteiller, E. Merck, U.A. Hasan, S. Hubac, B. Benguigui, G. Tinchieri, E.E.N. Bates, C. Caux, Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH, J. Biol. Chem. 280 (2005) 38133e38145. [9] S.J. DeWitte-Orr, K.L. Mossman, dsRNA and the innate antiviral immune response, Future Virol. 5 (3) (2010) 325. [10] S.J. DeWitte-Orr, J.C. Leong, N.C. Bols, Induction of antiviral genes, Mx and vig1, by dsRNA and chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines, Fish Shellfish Immunol. 23 (3) (2007) 670e682. [11] S.J. DeWitte-Orr, D.R. Mehta, S.E. Collins, M.S. Suthar, M. Gale, K.L. Mossman, Long double-stranded RNA induces an antiviral response independent of IFN regulatory factor 3, IFN-B promoter stimulator 1 and IFN, J. Immunol. 183 (10) (2009) 6545e6553.

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