Accepted Manuscript Characterization and function of GSK3β from Litopenaeus vannamei in WSSV infection Lingwei Ruan, Huachun Liu, Hong Shi PII: S1050-4648(18)30508-4 DOI: 10.1016/j.fsi.2018.08.032 Reference: YFSIM 5490 To appear in: Fish and Shellfish Immunology Received Date: 21 June 2018 Revised Date: 13 August 2018 Accepted Date: 16 August 2018 Please cite this article as: Ruan L, Liu H, Shi H, Characterization and function of GSK3β from Litopenaeus vannamei in WSSV infection, Fish and Shellfish Immunology (2018), doi: 10.1016/ j.fsi.2018.08.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Characterization and Function of GSK3β from Litopenaeus 2 vannamei in WSSV infection 3 Lingwei Ruana, *, Huachun Liua, b, Hong Shia 5 a 6 of Marine Genetic Resources of State Oceanic Administration, Third Institute of 7 Oceanography, State Oceanic Administration, Fujian Key Laboratory of Marine 8 Genetic Resources, South China Sea Bio-Resource Exploitation and Utilization 9 Collaborative Innovation Center, Xiamen 361005, People's Republic of China. RI PT 4 10 b 11 China. School of Life Science, Xiamen University, Xiamen 361005, People's Republic of 12 * Corresponding author: 14 Lingwei Ruan 15 Third Institute of Oceanography 16 No. 184 Daxue Road 17 Xiamen, Fujian 18 P. R. China (361005) 19 Tel: +86 592 2195856 20 Fax: +86 592 2195856 21 Email: email@example.com 23 AC C EP TE D 13 22 M AN U SC State Key Laboratory Breeding Base of Marine Genetic Resources, Key Laboratory ACCEPTED MANUSCRIPT 24 Abstract: GSK3β, a serine/threonine protein kinase, is a crucial regulator in several 26 signaling pathway and plays a vital role in multiple cellular processes including cell 27 proliferation, growth, apoptosis and immune response. In this study, a GSK3β 28 homolog from L. vannamei, designed as LvGSK3β, was characterized. Sequence 29 analysis showed that LvGSK3β possessed a highly similarity with GSK3β from other 30 species, which contained a catalytic domain and serine/threonine phosphorylation 31 sites. To analyze the role of LvGSK3β in the process of white spot syndrome virus 32 (WSSV) infection, real-time quantitative PCR and western blot assays were 33 performed. The results showed that the transcription and expression levels of 34 LvGSK3β were inhibited upon WSSV challenge, accompanied with down-regulated 35 phosphorylation levels. When LvGSK3β was silenced, the transcription of WSSV 36 gene ie1 was inhibited, and the apoptosis of hemocytes induced by WSSV was 37 up-regulated remarkably as well. In addition, inactivation of LvGSK3β could also 38 depress virus infection that further validated the results. Conclusively, LvGSK3β was 39 an important protein for shrimp immunomodulation, and shrimp might promote the 40 apoptosis to restrain WSSV infection by inhibition of LvGSK3β. The study will be 41 helpful for understanding the molecular mechanism of host-virus interaction. 44 45 SC M AN U TE D EP 43 Key words: Litopenaeus vannamei, WSSV, GSK3β, apoptosis AC C 42 RI PT 25 1. Introduction 46 As an important cultured shrimp, Litopenaeus vannamei is commonly reared 47 worldwide and contributes nearly 80% to world production of the total economic 48 penaeid shrimp . However, disease outbreaks resulted from diverse pathogens have 49 severely threatened shrimp aquaculture. Among them, white spot disease (WSD) 50 which caused by white spot syndrome virus (WSSV) is the greatest menace with high ACCEPTED MANUSCRIPT 51 mortality, and caused dramatic economic losses . Although considerable scientific 52 studies have been aimed for years, the knowledge about the host-virus interaction is 53 quite limited. Thus, it’s imperative to understand the molecular mechanisms involved 54 in immune defense to improve disease control. Innate immune system is critical for shrimp to defense against pathogens. 56 When suffered from pathogens, it can trigger a series of humoral immunity and 57 cellular immunity responses, which are always modulated by signaling transduction 58 [3, 4]. Many signaling pathways have been proved to be implicated in the process of 59 shrimp innate immune responses, such as Toll, NF-κB, PI3K/Akt, JAK/STAT and 60 Wnt/β-catenin [5-7]. Some components of these signaling pathways are critical to 61 host and virus, thus studies on these key molecules will be helpful for understanding 62 the regulations of shrimp innate immune. M AN U SC RI PT 55 Glycogen synthase kinase 3β (GSK3β) is a serine/threonine kinase with 64 originally function in glycogen biosynthesis . Subsequently it was discovered to be 65 a crucial regulator in many signaling pathways including Wnt/β-catenin, NF-κB and 66 PI3K/AKT, and participate in various physiological processes, such as cell 67 proliferation, differentiation, apoptosis and immune response [9, 10]. In the adaptive 68 immunity, GSK3β can modulate the clonal expansion and specificity of immune cells 69 by regulating their survival and differentiation directly or influencing the repertoire of 70 cytokines secretion indirectly . It has been found that the antigen-specific 71 stimulation of CD8+T cells could inactivate GSK3β to enhance the cytotoxic effects 72 . And GSK3β can regulate the differentiation of CD4+T cells into Th17 cells . 73 Recently, GSK3β has also been established as a key regulator of innate immune 74 system. In host antiviral responses, GSK3β could not only promote the production of 75 multiple cytokines (IFN-γ, IL-1β, TNF, IL-12 etc.) by activating STAT3, NF-κB, 76 AP-1 and other transcription factors, but also limit viral proliferation through binding 77 with a variety of viral protein, such as NS5A, LANA and HBX [11,14-18]. In addition, 78 there were several researches showed that GSK3β was also closely related to virus 79 infection. For example, coxsackievirus B3 (CVB3) could decrease the expression of AC C EP TE D 63 ACCEPTED MANUSCRIPT 80 β-catenin by activating GSK3β-dependent mechanism, thereby promoting cell 81 apoptosis and the release of virus particles . Thus it is implied that GSK3β might execute multiple functions in the host-virus 83 interaction. However, knowledge about its role in the invertebrate’s immune 84 regulation is little. In this study, a GSK3β homolog from L. vannamei, designed as 85 LvGSK3β, was characterized. And its role in the host-virus interaction in L. vannamei 86 was investigated. It was found that WSSV infection could down-regulate the 87 transcription and translation of LvGSK3β. Further studies revealed that silencing of 88 LvGSK3β could inhibit the viral gene transcription, accompanied by induction of cell 89 apoptosis. The results implied that LvGSK3β might be a key molecule involved in 90 immune regulation, and could helpfully improve our knowledge of the host-virus 91 interaction. 92 2. Materials and methods 94 2.1 Reagents and antibodies TE D 93 M AN U SC RI PT 82 The antibody against LvGSK3β was prepared in Xiamen University Laboratory 96 Animal Center by immunizing the mice with recombinant protein. Anti-p-GSK3β 97 (Ser9) was purchased from Cell Signaling Technology. Anti-p-GSK3β (Y216) and 98 anti-β-tubulin were bought from Sigma. Horseradish peroxidase (HRP)-conjugated 99 secondary antibodies were obtained from Thermo Scientific. GSK-3 Inhibitor IX was 101 102 AC C 100 EP 95 purchased from Merck Millipore 2.2 Shrimp culture and WSSV preparation 103 Healthy adult shrimp, L. vannamei, weight in 12-15g, were purchased from local 104 seafood market, and kept in air-pumped circulating seawater at 25°C for 3 days before 105 experiments. ACCEPTED MANUSCRIPT 106 107 WSSV used in the study was prepared from hemocytes of the viral infected crayfish and quantified as described before [20, 21]. 108 2.3 cDNA clone and sequence analysis RI PT 109 110 The full-length cDNA sequence of LvGSK3β, obtained from the L. vannamei 111 transcriptome, was cloned and deposited in NCBI database with accession no. 112 MG680178. The primers used for the PCR amplification was listed in Table1. The 113 sequence 114 (http://www.ncbi.nlm.nih.gov/BLAST/). The conserved domain within LvGSK3β was 115 predicted by the SMART program (http://smart.embl-heidelberg.de). Multiple 116 sequence alignment was performed using DNAMAN (Lynnon Biosoft), and the 117 phylogenic tree of GSK3β from various species was constructed by using Mega 6.06 118 with the Neighbor-Joining method (NJ). analysis LvGSk3β was analyzed by BLAST M AN U TE D 119 120 of SC homology 2.4 Total RNA extraction and reverse transcription PCR Total RNA originated from selected tissues of L. vannamei was extracted with 122 TRIzol reagent (Molecular Research Center) according to the instructions. After 123 digestion with DNaseI (Takara) at 37°C for 30 min, 2 µg of total RNA was used to 124 synthesis the first-strand cDNA by reverse transcriptase M-MLV (Takara) with 125 oligo(dT)18 primer (Fermentas) as described in the manuals. 127 AC C 126 EP 121 2.5 Real-time quantitative PCR 128 Real-time quantitative PCR was used for analyzing the mRNA levels of 129 LvGSK3β. Selected tissues from three individual shrimp were collected. Total RNA 130 extraction and cDNA synthesis were performed as described above. Rotor-Gene™ 131 6000 (Corbett Life Science) was used to perform real-time quantitative PCR assay. ACCEPTED MANUSCRIPT 132 The PCR procedure was as follows, 1 cycle of 95°C for 1 min, 40 cycles of 95°C for 133 10 s, 56°C for 15 s, and 72°C for 15 s. Lvtubulin was used as the reference control 134 and results were calculated with 2-∆∆CT methods. The primers used were listed in 135 Table 1. 137 RI PT 136 2.6 Tissue distribution analysis Total RNAs were isolated from various tissues including hemocyte, gill, heart, 139 hepatopancreas, muscle and intestine, according to the protocol mentioned above. 140 Then the first-strand cDNA was synthesized and real-time quantitative PCR was 141 performed with the primer pairs LvGSK3β-F2/LvGSK3β-R2 (Table 1). M AN U SC 138 142 143 2.7 Analysis of transcription post WSSV infection Healthy adult shrimps were cultured under laboratory environment for 3 days. 145 Then each shrimp was injected with 1×107 WSSV particles diluted in 100 µL PBS 146 (140 mM NaCl, 3 mM KCl, 8 mMNa2HPO4, 1.5 mM KH2PO4, pH 7.4), and shrimps 147 injected with 100 µL PBS were served as control. Gills and hemocytes from three 148 individual shrimp were sampled at 0, 6, 12, 24, 48 and 72 h post injection (hpi), 149 respectively. Thereafter the total RNAs were isolated, and transcriptional levels were 150 analyzed by real-time quantitative PCR. 152 153 EP AC C 151 TE D 144 2.8 Western blot analysis The shrimps were challenged as mentioned in section 2.7. Hemocytes collected 154 from WSSV-injected shrimps or PBS-injected shrimps were lysed with RIPA lysis 155 buffer (Beyotime) containing PMSF (1:100), protease inhibitor (Calbiochem, 1:200) 156 and phosphatase inhibitor (Merck Millipore, 1:50) on ice for 30 mins. The 157 supernatants of cell lysate were boiled for 7 mins mixed with 5×loading buffer (250 ACCEPTED MANUSCRIPT mM Tris-HCl pH 6.8, 10% SDS, 50% glycerol, 5% β-mercaptoethanol, 0.5% 159 bromophenol blue). Then the samples were separated by SDS-PAGE and transferred 160 onto PVDF membranes (GE Healthcare). After blocked with 5% (w/v) skim milk or 161 BSA resolved in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) for 162 1h at room temperature (RT), the membranes were incubated with corresponding 163 primary antibodies at 4°C with gentle shaking overnight. Thereafter, the membranes 164 were washed with TBST for 3 times and subsequently incubated with 165 HRP-conjugated secondary antibody for 1h at RT. The signals on the membranes 166 were detected by using Super-Signal West Pico Chemiluminescent (ECL) Substrates 167 (Thermo Scientific). SC RI PT 158 169 M AN U 168 2.9 RNAi assays Silencing the transcription of LvGSK3β was performed to explore its role in the 171 innate immune of shrimp in vivo. The LvGSK3β-dsRNA and eGFP (enhanced Green 172 Fluorescent Protein)-dsRNA (control) were synthesized according to the instructions 173 by using T7 RiboMAXTM express RNAi system (Promega). The primers designed for 174 dsRNA synthesis were listed in Table1. TE D 170 For RNAi assays, dsRNA (20 µg) diluted with PBS was intramuscularly injected 176 into abdominal segment of shrimps. And there was a twice injection with the 177 corresponding dsRNA after 24h to ensure the RNAi efficiency. After 12h post the 178 second injection, shrimps were challenged with 1×107 WSSV diluted in 100 µL PBS, 179 and the shrimps injected with 100 µL PBS were also served as control. Hemocytes 180 were collected at 1, 9, 24 hpi. Then the transcriptional levels of LvGSK3β and WSSV 181 immediate-early (IE) gene ie1 were detected by real-time quantitative PCR. AC C EP 175 182 183 2.10 Detection of caspase3/7 activity ACCEPTED MANUSCRIPT RNAi was performed as mentioned above and the hemocytes with different 185 treatments were collected. Portions of hemocytes were used to determine the 186 transcriptional levels of LvGSK3β and ie1. The remaining was used to analyze the 187 cell apoptosis by Caspase-Glo®3/7 Assay (Promega). The details were shown as 188 follows: 4×104 shrimp hemocytes were mixed with 50 µl Caspase-Glo®3/7, and 189 incubated at RT in the dark. After 1h, the mixture was analyzed by GloMax20/20 190 Luminometer (Promega) to detect the activity of caspase3/7. A paired Student’s t-test 191 was used to statistically analyze the data. 2.11 Shrimp hemocytes culture and inhibitor treatment M AN U 193 SC 192 RI PT 184 Hemocytes from L. vannamei were extracted with addition of anticoagulants (26 195 mM sodium citrate, 100 mM glucose, 140 mM NaCl, 30 mM citric acid, pH6.0), and 196 cultivated in cell culture dishes for 45 min at 27℃. Adherent cells were washed 197 carefully with PBS before seeded in L15 medium (Gibco) supplemented with 15% 198 FBS (Gibco), penicillin (60 µg/mL) and streptomycin (50 µg/mL). 5 µM GSK-3 199 Inhibitor IX or DMSO (control) were added. Firstly, Cell Counting Kit-8 (CCK-8, 200 Dojindo) was used to determine the cytotoxicity of the reagents on cells according to 201 the instructions. Then western blot was applied to detect the effect of GSK-3 Inhibitor 202 IX on LvGSK3β. After being pretreated with reagents for 1h, the hemocytes were 203 incubated with 1×107 WSSV particles diluted in L15 medium for another 5h. 204 Subsequently, the samples were collected and the total RNA extraction and reverse 205 transcription were achieved by Single Cell-to-CT™ qRT-PCR Kit (Ambio). Finally, 206 the synthetic cDNA was applied to analyze the transcription of ie1 using real-time 207 quantitative PCR AC C EP TE D 194 208 209 3 Results 210 3.1 Cloning and sequence analysis of LvGSK3β ACCEPTED MANUSCRIPT The cDNA sequence of LvGSK3β contained an open reading frame (ORF) of 212 1233 bp, which encoded 410 amino acid residues with a calculated molecular weight 213 of 46.029 KDa (Fig. 1A). SMART analysis displayed that LvGSK3β protein had 214 characteristic catalytic domain of serine/threonine protein kinase from residues 54 to 215 338, namely protein kinase domain (PKD) (Fig. 1A). Multiple sequence alignment 216 revealed that LvGSK3β shared 88.78%, 77.38%, 77.29%, 76.21% and 75% identity to 217 Eriocheir sinensis GSK3β (EsGSK3β), Branchiostoma floridae GSK3β (BfGSK3β), 218 Cerapachys biroi GSK3β (CbGSK3β), Picoides pubescens GSK3β (PpGSK3β) and 219 Homo sapiens GSK3β (HsGSK3β), respectively, suggesting that GSK3β proteins 220 were highly conserved from invertebrates to vertebrates (Fig. 1B). In addition, we 221 found that the LvGSK3β Ser9 and Tyr214 phosphorylation residues were respectively 222 corresponded to the Ser9 and Tyr216 residues of GSK3β in mammalian (Fig. 1B). 223 Furthermore, phylogenetic analysis showed that LvGSK3β was clustered together 224 with arthropods (Fig. 1C). 226 TE D 225 M AN U SC RI PT 211 3.2 Tissue distribution of LvGSK3β To analyze the tissue distribution of LvGSK3β, total RNA of the hemocytes, gill, 228 heart, hepatopancreas, muscle and intestine were extracted and reversely transcribed 229 into cDNA for real-time quantitative PCR assays. As shown in Fig. 2, transcripts of 230 LvGSK3β could be detected in all selected tissues, with a highest level in heart. 232 233 AC C 231 EP 227 3.3 Transcription analysis of LvGSK3β post WSSV infection In order to investigate the relationship between LvGSK3β and WSSV infection, 234 hemocytes and gills from shrimp challenged with WSSV were collected at different 235 time points post WSSV infection, and the total RNA was isolated for cDNA synthesis. 236 Then the transcriptional profiles of LvGSK3β were analyzed by real-time quantitative 237 PCR. WSSV IE genes have important regulatory roles in the process of viral infection ACCEPTED MANUSCRIPT and proliferation. And the transcriptional level of ie1, a well-recognized IE gene of 239 WSSV, was used as an index for determining the infection of WSSV. As shown in 240 Fig. 3A, C, the transcription of ie1 increased gradually with lastingness of infection, 241 indicating WSSV infected successfully in shrimp. Then the transcription profiles of 242 LvGSK3β were analyzed. In hemocytes, it was not significantly changed at the early 243 stage of WSSV infection (6h), and decreased with the virus infection (12h-72h) (Fig. 244 3B). Similarly, WSSV challenge also had inhibited effects on LvGSK3β transcription 245 in gill before 48h post challenge (Fig. 3D). Taken together, WSSV infection could 246 down-regulate the transcription of LvGSK3β. SC M AN U 247 248 RI PT 238 3.4 Expression and phosphorylation level of LvGSK3β upon WSSV challenge To further study whether WSSV infection can affect the translation of LvGSK3β, 250 shrimps were intramuscularly injected with WSSV or PBS as mentioned above. 251 Hemocytes lysates were prepared at different time points post injection. Then western 252 blot analysis was performed, and the results showed that WSSV infection could also 253 significantly reduce the total protein level of LvGSK3β in shrimp, while no much 254 improvement showed in control group (Fig. 4A, B). As a serine/threonine kinase, the 255 activity of GSK3β is mainly regulated by its phosphorylation. Furthermore, the 256 phosphorylation of Ser9 and Tyr214 residues were also tested. The results displayed 257 that the phosphorylation of LvGSK3β at these two residues were both prominently 258 down-regulated after WSSV challenge (Fig. 4A). In sum, the results displayed that the 259 transcription, translation and phosphorylation of LvGSK3β were all down-regulated 260 by WSSV infection, suggesting LvGSK3β had close relationship with WSSV 261 infection. AC C EP TE D 249 262 263 264 3.5 Knockdown of LvGSK3β inhibited WSSV infection by promoting hemocytes apoptosis ACCEPTED MANUSCRIPT To further investigate the role of LvGSK3β in the process of WSSV infection, 266 RNAi was performed to silence LvGSK3β in vivo. Firstly, shrimps were injected with 267 dsLvGSK3β, and the knock-down efficiency of LvGSK3β was detected using 268 real-time quantitative PCR. The results displayed that the transcriptional levels of 269 LvGSK3β was significantly repressed in dsLvGSK3β-treated shrimps, while there 270 was no obvious changes in the control groups, which were injected with dseGFP (Fig. 271 5A, D). Upon WSSV stimulated, silencing of LvGSK3β would remarkably inhibit the 272 transcription level of ie1 at 9 hpi and 24 hpi (Fig. 5B), indicating that WSSV infection 273 was restrained in shrimp. Moreover, the hemocytes apoptosis was determined by 274 testing the caspase3/7 activity. Compared with dseGFP group, silence of LvGSK3β 275 could significantly increase caspase3/7 activity of shrimp hemocytes at the early stage 276 of WSSV infection (Fig. 5C), while it was repressed with no virus stimulation (Fig. 277 5E). These data gave a clue that LvGSK3β had an important role in WSSV infection, 278 and shrimp might restrain the virus infection through inducing cell apoptosis, which 279 was mediated by inhibiting the expression of LvGSK3β expression. 281 SC M AN U TE D 280 RI PT 265 3.6 Effects of the LvGSK3β on virus infection GSK3β functions as a phosphorylated kinase to regulate multiple substrates in 283 mammalians. In the study, GSK-3 Inhibitor IX, an effective and specific 284 ATP-competitive inhibitor of GSK3β, was used to inhibit the LvGSK3β activity to 285 further explore the relationship between LvGSK3β and WSSV infection. Firstly, 286 cytotoxicity test using CCK-8 was performed, and the results displayed that the 287 reagent had little effect on hemocytes. The survival rate of cells treated with GSK-3 288 Inhibitor IX (5 µM) were more than 80%, which was similar to those treated with 289 DMSO (control) (Fig. 6A). Then the effect of GSK-3 Inhibitor IX was validated by 290 western blot, and it showed that GSK-3 Inhibitor IX could really inhibit the 291 phosphorylation of LvGSK3β (Fig. 6B). Furthermore, the role of LvGSK3β on 292 WSSV infection was investigated. Comparing to the control group, the primary 293 hemocytes pretreated with GSK-3 Inhibitor IX could repress the transcription of ie1 AC C EP 282 ACCEPTED MANUSCRIPT 294 (Fig. 6C). The results were consistent with that of RNAi, implied that LvGSK3β 295 might be necessary for WSSV infection. 296 Discussion RI PT 297 Cell signaling transduction pathway plays an important role in the regulation of 299 shrimp antiviral immune response, including Wnt/β-catenin, JAK-STAT, PI3K/Akt 300 and NF-kB. Moreover, increasing evidences have found that several key molecules, 301 such as NF-kB, β-catenin, AP-1 and STAT, are key modulated points in the 302 shrimp-WSSV interaction. As a highly conserved multifunctional serine/threonine 303 kinase, GSK3β is broadly involved in many cellular functions, particularly in 304 regulating many components of the innate immune response. Most of studies about 305 innate immune mediated by GSK3β were focused on vertebrates, while the 306 knowledge about its role in regulating innate immunity of invertebrates is limited. M AN U SC 298 In the present study, we cloned and characterized a GSK3β homolog, named as 308 LvGSK3β, from L. vannamei. Sequence analysis displayed that LvGSK3β shared a 309 high similarity with identified GSK3β from other species and contained a conserved 310 protein kinase domain (PKD), which is the characteristic of serine/threonine protein 311 kinases family. By multiple sequence alignment, we found that LvGSK3β also had 312 Ser9 and Tyr214 phosphorylation sites, which were corresponded to Ser9 and Tyr216 313 phosphorylation sites of GSK3β from mammalian, respectively. The results suggested 314 that LvGSK3β belonged to the GSK3 family, and might share similar biological 315 functions with other known GSK3β. EP AC C 316 TE D 307 In order to reveal the function of LvGSK3β in the shrimp-WSSV interaction, the 317 transcriptional and translational profiles of LvGSK3β post WSSV infection were 318 firstly analyzed by real-time quantitative PCR and western blot. It was shown that 319 WSSV infection could decrease the transcription and expression levels of LvGSK3β, 320 accompanied with down-regulating the phosphorylation of Ser9 and Tyr214 sites (Fig. ACCEPTED MANUSCRIPT 3, 4). These data implied that LvGSK3β had a close relationship with WSSV infection. 322 Then the role of LvGSK3β was further investigated to determine whether LvGSK3β 323 is beneficial to immunomodulation. RNAi assays were performed to silence the 324 expression of LvGSK3β. Meanwhile, an effective and specific inhibitor of GSK3β, 325 GSK-3 Inhibitor IX, was used to inactivate LvGSK3β. Both of the results showed that 326 once LvGSK3β was down-regulated, WSSV infection could be repressed (Fig. 5, 6). 327 Moreover, cell apoptosis induced by WSSV could be promoted significantly. The 328 results suggested that LvGSK3β was an important molecule for antiviral immune 329 regulation in shrimp, which might mediate apoptosis to facilitate the clearance of 330 WSSV. In the study of influenza infection, similar results were presented. 331 Down-regulation of GSK3β expression could also positively regulated the 332 virus-induced host cell apoptosis to inhibit replication of influenza virus . M AN U SC RI PT 321 In previous study, we have found that WSSV infection could promote the 334 ubiquitination of Lvβ-catenin, which resulted in restrictions on nuclear translocation. 335 When the protein levels of Lvβ-catenin were up-regulated by GSK-3 Inhibitor IX, 336 WSSV infection was also significantly inhibited , which was consistent with the 337 results. Phosphorylation by GSK3β on β-catenin is the switch of the degradation of 338 β-catenin mediated by ubiquitination. It is reasonable that shrimp might inactivate 339 LvGSK3β to repress the ubiquitination and promote the nuclear translocation of 340 Lvβ-catenin for immune regulation. As GSK3β is a cross point in several signal 341 pathway, and it has extensive substrates, the specific regulation mechanism of 342 LvGSK3β on the antiviral immune response of shrimp needs to be further studied. EP AC C 343 TE D 333 In sum, a novel GSK3β homolog was identified in L. vannamei, which had a 344 close relationship with WSSV infection. Further study revealed that silencing the 345 transcription or inactivation of LvGSK3β could repress WSSV infection. The process 346 might be mediated by promoting the cell apoptosis induced by WSSV. This study will 347 be helpful to clarify the molecular mechanism of host-virus interaction and to advance 348 the control of shrimp disease. 349 ACCEPTED MANUSCRIPT 350 Acknowledgements This work was funded by the National Natural Science Foundation of China (No. 352 31472297), the China Agriculture Research System-48, and Natural Science 353 Foundation of Fujian Province of China (2018J01048). RI PT 351 354 References 356 1. 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 M AN U 360 TE D 359 EP 358 Briggs M, Fungesmith S, Subasinghe RP, Phillips M. Introductions and movement of two penaeid shrimp species in Asia and the Pacific. Fao Fisheries Technical Paper. 2005. 2. Lightner DV. Virus diseases of farmed shrimp in the Western Hemisphere (the Americas): a review. Journal of Invertebrate Pathology. 2011 106:110-30. 3. Borregaard N, Elsbach P, Ganz T, Garred P, Svejgaard A. Innate immunity: from plants to humans. Immunology Today. 2000 21:68. 4. Hoffmann JA, Kafatos FC, Janeway CA, Ezekowitz RAB. Phylogenetic Perspectives in Innate Immunity. Science. 1999 284:1313-8. 5. Li F, Xiang J. Signaling pathways regulating innate immune responses in shrimp. Fish & Shellfish Immunology. 2013 34:973-80. 6. Song X, Zhang Z, Wang S, Li H, Zuo H, Xu X, et al. A Janus Kinase in the JAK/STAT signaling pathway from Litopenaeus vannamei is involved in antiviral immune response. Fish Shellfish Immunol. 2015 44:662-73. 7. Xie YK, Ding D, Wang HM, Kang CJ. A homologue gene of β-catenin participates in the development of shrimps and immune response to bacteria and viruses. Fish & Shellfish Immunology. 2015 47:147-56. 8. Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. FEBS Journal. 2005 107:519-27. 9. Forde JE, Dale TC. Glycogen synthase kinase 3: a key regulator of cellular fate. Cellular & Molecular Life Sciences Cmls. 2007 64:1930. 10. Jope RS, Yuskaitis CJ, Beurel E. Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics. Neurochemical Research. 2007 32:577-95. 11. Beurel E, Michalek SM, Jope RS. Innate and adaptive immune responses regulated by glycogen synthase kinase-3 (GSK3). Trends in Immunology. 2010 31:24-31. AC C 357 SC 355 ACCEPTED MANUSCRIPT 421 Table 1 422 Primers used in the study 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 SC 389 M AN U 388 TE D 387 EP 386 AC C 385 RI PT 420 12. Ohteki T, Parsons M, Zakarian A, Jones RG, Nguyen LT, Woodgett JR, et al. Negative Regulation of T Cell Proliferation and Interleukin 2 Production by the Serine Threonine Kinase Gsk-3. Journal of Experimental Medicine. 2000 192:99-104. 13. Beurel E, Yeh WI, Michalek SM, Harrington LE, Jope RS. Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells. Journal of Immunology. 2011 186:1391-8. 14. Martin M, Rehani K, Jope RS, Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nature Immunology. 2005 6:777-84. 15. Jope RS, Cheng Y, Lowell JA, Worthen RJ, Sitbon YH, Beurel E. Stressed and Inflamed, Can GSK3 Be Blamed? Trends in Biochemical Sciences. 2016. 16. Park CY, Choi SH, Kang SM, Ahn BY, Kim H, Jung G, et al. Nonstructural 5A protein activates beta-catenin signaling cascades: implication of hepatitis C virus-induced liver pathogenesis. Journal of Hepatology. 2009 51:853-64. 17. Liu J, Martin HJ, Shamay M, Woodard C, Tang QQ, Hayward SD. Kaposi's Sarcoma-Associated Herpesvirus LANA Protein Downregulates Nuclear Glycogen Synthase Kinase 3 Activity and Consequently Blocks Differentiation. Journal of Virology. 2007 81:4722-31. 18. Kuo CY, Wu CC, Hsu SL, Hwang GY. HBx Inhibits the growth of CCL13-HBX-stable cells via the GSK-3beta/beta-catenin cascade. Intervirology. 2008 51:130-6. 19. Yuan J, Zhang J, Wong BW, Si X, Wong J, Yang D, et al. Inhibition of glycogen synthase kinase 3beta suppresses coxsackievirus-induced cytopathic effect and apoptosis via stabilization of beta-catenin. Cell Death & Differentiation. 2005 12:1097. 20. Xie X, Li H, Xu L, Yang F. A simple and efficient method for purification of intact white spot syndrome virus (WSSV) viral particles. Virus Research. 2005 108:63. 21. Zhou Q, Qi YP, Yang F. Application of spectrophotometry to evaluate the concentration of purified White Spot Syndrome Virus. Journal of Virological Methods. 2007 146:288-92. 22. Dai X, Zhang L, Han H. The Impact of GSK-3β on the Replication of Influenza Virus in A549 Cells. Open Journal of Nature Science. 2016 04:371-7. 23. Sun J, Ruan L, Shi H, Xu X. Characterization and function of a β-catenin homolog from Litopenaeus vannamei in WSSV infection. Developmental & Comparative Immunology. 2017 76:412. 384 ACCEPTED MANUSCRIPT Sequence (5’-3’) Name For LvGSK3β cDNA clone LvGSK3β-F1 TCCTCCTTATTTGAACCTTTCCGAC LvGSK3β-R1 GGCAGGAAGGCTGGTGTGGAG GTGTGGACCAGTTAGTAG LvGSK3β-R2 CCAGGTTTATAGCGTCTTC ie1-F GCACAACAACAGACCCTACCC ie1-R GAAATACGACATAGCACCTCCAC Lvtubulin-F GCCTCGTGCCATCCTTGTTG Lvtubulin-R CCCTTAGCCCAGTTGTTTCCAG SC LvGSK3β-F2 For dsRNA templates amplification dsRNA-LvGSK3β-R GAACACCTTCTGCCATGGAT dsRNA-LvGSK3β-T7-F a GGATCCTAATACGACTCACTATAGGAGGTGCTTCAGGACAAACGCTT dsRNA-LvGSK3β-T7-R a GGATCCTAATACGACTCACTATAGGGAACACCTTCTGCCATGGAT dsRNA-eGFP-F GTGCCCATCCTGGTCGAGCT dsRNA-eGFP-R TGCACGCTGCCGTCCTCGAT dsRNA- eGFP -T7-F a GGATCCTAATACGACTCACTATAGGGTGCCCATCCTGGTCGAGCT dsRNA- eGFP -T7-R a GGATCCTAATACGACTCACTATAGGTGCACGCTGCCGTCCTCGAT a The sequences of T7 promoter were underlined 424 TE D 423 AGGTGCTTCAGGACAAACGCTT M AN U dsRNA-LvGSK3β-F RI PT For real-time quantitative PCR Figure Legends 426 Fig. 1. Sequence analysis of LvGSK3β. (A) The nucleotide and deduced amino acid 427 sequences of LvGSK3β. The gray-shade region indicated the serine/tyrosine kinase 428 domain (54-338a). The predicted phosphorylation sites were indicated with 429 black-shade. (B) Multiple sequence alignment among GSK3β from L. vannamei and 430 other species. The conserved phosphorylated sites were marked with red star. (C) 431 Phylogenetic tree analysis of GSK3β from L. vannamei and other species. Genbank 432 accession number of each GSK3β was shown after their scientific names. LvGSK3β 433 was marked with a black dot. AC C EP 425 ACCEPTED MANUSCRIPT Fig. 2. Tissue distribution analysis of LvGSK3β by real-time quantitative PCR in L. 435 vannamei. Lvtubulin was used as internal reference. 436 Fig. 3. Transcriptional profile of LvGSK3β post WSSV infection. (A) Transcriptional 437 level of ie1 after WSSV challenge in hemocytes. (B) Transcriptional level of 438 LvGSK3β after injected with WSSV or PBS in hemocytes. (C) Transcriptional level 439 of ie1 after WSSV challenge in gill. (D) Transcriptional level of LvGSK3β after 440 injected with WSSV or PBS in gill. The tissues were collected at indicated time post 441 injection for analysis. The values were normalized to shrimp tubulin by using 2-△△CT 442 methods and the data were presented as means ± SD of triplicate experiments. The 443 statistical significance was calculated using Student’s t-test (*p < 0.05, **p < 0.01). 444 Fig. 4. Expression and phosphorylation levels of LvGSK3β upon WSSV challenge. (A) 445 Expression and phosphorylation profiles of LvGSK3β post WSSV challenge. (B) 446 Expression and phosphorylation profiles of LvGSK3β post PBS injection. Western 447 blot analysis with antibodies against LvGSK3β, p-LvGSK3β (Ser9), p-LvGSK3β 448 (Tyr214) was performed. Lvtubulin was used as internal control. 449 Fig. 5. Effect of dsRNA-mediated LvGSK3β silencing on WSSV infection and the 450 shrimp hemocytes apoptosis. (A) Efficiency of dsGSK3β silencing in shrimps post 451 WSSV infection. Real-time quantitative PCR was used, and the dseGFP was served as 452 control. (B) Transcription of ie1 in dsRNA-treated shrimps post WSSV infection. (C) 453 Analysis of the hemocytes apoptosis with dsGSK3β or dseGFP treatment post WSSV 454 infection. (D) Efficiency of dsGSK3β silencing in shrimps post PBS injection. 455 Real-time quantitative PCR was used, and the dseGFP was served as control. (E) 456 Analysis of the hemocytes apoptosis with dsGSK3β or dseGFP treatment post 457 injection with PBS. 458 Fig. 6. Effect of LvGSK3β inactivation on WSSV infection. (A) Analysis of relative 459 survival rate. Hemocytes were treated with GSK-3 inhibitor IX (5 µM) or DMSO 460 (control), and the cytotoxicity of the reagents was analyzed with Cell Counting Kit-8. 461 (B) Detection of LvGSKβ in the primary shrimp hemocytes with different treatments. AC C EP TE D M AN U SC RI PT 434 ACCEPTED MANUSCRIPT The hemocytes treated with GSK-3 inhibitor IX (5 µM) or DMSO (control) for 2h 463 were collected and analyzed by western blot. (C) Effects of GSK-3 inhibitor IX 464 treatment on WSSV gene transcription. After treatment with GSK-3 inhibitor IX (5 465 µM) or DMSO (control) for 1h, 1×107 WSSV virus particles were added and 466 incubated for 5h. Real-time quantitative PCR were used for analyzing the 467 transcription profile of ie1. AC C EP TE D M AN U SC RI PT 462 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT Highlights A GSK3β homologue of Litopenaeus vannamei was characterized. The transcription and expression of LvGSK3β were regulated by WSSV RI PT infection. AC C EP TE D M AN U SC LvGSK3β could regulate the apoptosis to restrain WSSV infection.