Protein Phosphatase PP1 Negatively Regulates IRF3 in Response to GCRV Infection in Grass Carp (Ctenopharyngodon idella)

Protein phosphatase-1 (PP1) has an important role in many cell functions, such as cell differentiation, development, immune response and tumorigenesis. However, the specific role of PP1 in the antiviral response in fish remains to be elucidated. In this study, the PPP1R3G homolog was identified in the grass carp (Ctenopharyngodon idella) and its role in defence against the GCRV infection was investigated. Phylogenetic analysis demonstrated that CiPPP1R3G clustered with homologues from other teleosts. Temporal expression analysis in vivo revealed that the expression level of CiPPP1R3G was significantly up-regulated in response to GCRV infection in grass carps, especially in the intestine and head-kidney. Cellular distribution analysis revealed that CiPPP1R3G was located in the nucleus and cytoplasm. Overexpression of CiPPP1R3G significantly negatively regulated the expression of CiIRF3, thus inhibiting its activation. In summary, we systematically analyzed the PPP1R3G gene in grass carp and illustrated its function as a negative regulator in the anti-GCRV immune responses.


INTRODUCTION
Protein phosphatase 1 (PP1), a serine (Ser)/threonine (Thr) phosphatase, is a member of the phosphoprotein phosphatase (PPP) superfamily (1)(2)(3). It is one of the most conserved proteins in eukaryotic cells (4). PP1 plays crucial roles in many biological processes including cell division and meiosis, metabolism, cell cycle arrest and apoptosis. It exerts these functions through the nucleophilic attack to catalyse the hydrolysis of serine/threoninelinked phosphate monoesters, and then dephosphorylates the substrate (4,5). PP1 is composed of a glycogen-targeting regulatory (G) subunit and a catalytic subunit (PP1c) (4). These regulatory subunits are very important for the function of PP1, and seven genes that encode different G subunits have been recognised so far: PPP1R3A to PPP1R3G (4).
PPP1R3G, as a regulatory subunit of PP1, was demonstrated to be involved in the regulation of glucose homeostasis and hepatic glycogenesis in mice (6). Another study reported that the AKT (serine-threonine protein kinase) directly phosphorylated the PPP1R3G in response to insulin or feeding in hepatocytes, and the phosphorylation of PPP1R3G accelerates postprandial glucose clearance and glycogenesis (7).
However, the function of PPP1R3G in the innate immunity still remains to be elucidated. Innate immunity is the fundamental defence system to protect animals from the infection of invading pathogens (8). In the antiviral immune response process, viral nucleic acids can be recognized by some pattern recognition receptors (PRRs), such as the NOD-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and Toll-like receptors (TLRs) (9)(10)(11)(12)(13)(14).
Grass carp is an economically important fish widely cultured in more than 40 countries (15). Its annual production reached 5.53 million tons in 2019, accounting for 18.36% of the harvest of all freshwater Chinese fisheries in that year (16). Nevertheless, its cultivation industry is often plagued by frequent outbreaks of hemorrhagic disease (17)(18)(19), caused by the grass carp reovirus (GCRV), a double-stranded RNA (dsRNA) virus belonging to the family Reoviridae, genus Aquareovirus (20).
In this study, we identified and investigated the PPP1R3G gene in grass carp (CiPPP1R3G). Our data revealed that PPP1R3G was up-regulated at the mRNA level after the GCRV infection in grass carp and demonstrated that it is a negative regulator in the anti-GCRV immune responses.

Experimental Fish
Healthy full-sib 3-months grass carps weighing ∼30 g were used in the study. The fish were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences and maintained in aerated freshwater at 26-28°C. All animal experiments were approved by the Animal Research and Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.

Cells and Viruses
Grass carp kidney (CIK) cells were maintained at 28°C in the medium 199 (Invitrogen) supplemented with 15% foetal bovine serum (FBS, Invitrogen). Human embryonic kidney (HEK) 293T cells were grown at 37°C and 5% CO 2 in a DMEM medium (Invitrogen) supplemented with 15% FBS. Type I grass carp reovirus (GCRV-0901, 10 6 TCID50/ml) was propagated in CIK cells until cytopathic effects (CPE) were observed, and then the cultured media with cells were harvested and stored at −80°C until use. Type II grass carp reovirus (GCRV-HZ08) was diluted to the titer of 2.97×10 3 RNA copy/ml for use in the experiments.

Identification and Sequence Analysis of CiPPP1R3G
According to the previous study of the transcriptome of the grass carp (21), the CiPPP1R3G mRNA (GenBank accession number MT833843) was obtained by PCR amplification using the primers listed in Table 1. The searches for similar protein sequences were performed by the BLASTP program, and conserved domain features were predicted using the SMART program (http://smart.emblheidelberg.de/). The phylogenetic tree was constructed using the Neighbour-joining method (NJ) with 1000 bootstraps in MEGA 7.0.

Experimental Viral Infection, Sample Collection, and Histological Observation
Healthy full-sib 3-month-old grass carps were divided into two groups: the GCRV-treated group and negative control group (approximately 150 specimens per group). Each fish in the experimental group (I) was infected via an intraperitoneal injection of 200 ml of GCRV-HZ08 (2.97 × 10 3 RNA copy/ml), while fish from the control group (II) were injected with 200 ml PBS. At 1-7 days post-injection, samples of spleen, liver, intestine, head-kidney and muscle tissues were harvested from both groups (biological replicates: n=4 specimens from each group). For the histological examination analyses, the intestine samples of both GCRV-treated (3-days post infection) and control groups (n=3, respectively) were fixed in the formalin, sectioned, stained with hematoxylin and eosin, and analyzed by two pathologists independently using light microscopy.

Quantitative Real-Time PCR and Tissue Expression of CiPPP1R3G
The total RNA of cells and spleen, liver, intestine, muscle and head-kidney tissues were extracted from four randomly selected grass carp specimens 1-4 days post GCRV infection, whereas 5-7 days post GCRV infection specimens exhibiting typical symptoms of disease (e.g., muscle bleeding) were selected. After grinding each sample in liquid nitrogen, 1 ml of Trizol reagent was added per 50 mg of tissue.  Table 1. We tested the suitability of three commonly used internal reference genes, including b-actin, GAPDH and B2M, all of which exhibited a relatively stable expression in a previous study (22). Their expression was studied by qRT-PCR, and results analysed using the NormFinder software (22

Plasmid Construction
The open reading frame (ORF) of CiPPP1R3G was subcloned into pCMV-Flag vector (Clontech) and pAcGFP-N1 (Clontech). The ORF of grass carp IRF3 (KC898261) was subcloned into pCMV-Myc vector (Clontech) and pM-RFP vector (Clontech). The grass carp IFN (CiIFN) was obtained from the lab of Prof. Su (Huazhong Agriculture University, China) and cloned into pGL3-Basic luciferase reporter vector (Promega). The plasmid containing CiIFN1pro-Luc in pGL3-Basic luciferase reporter vectors were constructed as described previously (24). All recombinant plasmids were verified by DNA sequencing.

Transient Transfection and Virus Infection
For the luciferase assay, transient cell transfections were performed in CIK cells seeded in 6-well or 24-well plates by using Lipofectamine 2000 Transfection Reagent (Invitrogen) according to the manufacturer's protocol. CIK cells were seeded in 6-well plates overnight and transfected with CiIRF3-Myc or the pCMV-Myc vector separately (2 µg/well for luciferase analysis and 1 µg/well for qRT-PCR). Next, to investigate the response of CiPPP1R3G to CiIRF3, CIK cells were seeded in 6well plates overnight and transfected with CiPPP1R3G-Flag, CiIRF3-Myc and the pCMV-Myc vector together (1 µg/well for qRT-PCR). The virus titration (GCRV-0901) was examined in CIK cells as previously described (25).

Reporter Gene Analysis
To investigate the interferon promoter activity evoked by the CiPPP1R3G, pGL3-basic luciferase reporter vector and CiIFN1-Luc (obtained from the lab of Prof. Su) were co-transfected in CIK cells as described previously (26). The pRL-CMV (Promega) plasmid was co-transfected to normalize the transfection efficiencies. CIK cells were seeded into 24-well plates, and 24 h later co-transfected with: 1) 300 ng CiIFN1-pro-Luc plasmid, 300 ng pCMV-Myc plasmid and 300 ng pCMV-Flag plasmid; 2) 300 ng CiIFN1pro-Luc plasmid, 300 ng pCMV-Myc plasmid and 300 ng CiPPP1R3G-Flag plasmid; 3) 300 ng CiIFN1pro-Luc plasmid, 300 ng CiIRF3-Myc plasmid and 300 ng pCMV-Flag plasmid; 4) 300 ng CiIFN1pro-Luc plasmid, 300 ng CiIRF3-Myc plasmid and 300 ng CiPPP1R3G-Flag plasmid. Renilla luciferase internal control vector (30 ng, pRL-CMV, Promega) was added in each group. At 24 h post-transfection, the cells were washed in PBS and lysed to measure the luciferase activity by Dual-Luciferase Reporter Assay System, according to the manufacturer's protocol (Promega). Firefly luciferase activities were normalized on the basis of Renilla luciferase activity. The final results were calculated as averages of more than three independent experiments, each performed in triplicate.

Subcellular Localization Analysis
To investigate the subcellular localization of CiPPP1R3G and CiIRF3, GFP-fused CiPPP1R3G (pAcGFP-N1-CiPPP1R3G) vector plasmids and RFP-fused CiIRF3 (pAcRFP-N1-CiIRF3) vector plasmids were transfected into CIK cells. CIK cells were plated onto coverslips in confocal dishes and transfected with the above plasmids for 24h. Then the cells were washed twice with PBS and fixed with 4% PFA for 1h. Finally, the samples were visualized using a laser scanning confocal microscope (Carl Zeiss).

Co-IP Analysis
For transient-transfection and Co-IP experiments, HEK293T cells were seeded into two petri dishes (10 cm in diameter) overnight and then cotransfected with a total of 10 µg of the following plasmids: (1) CiPPP1R3G-Flag and CiIRF3-Myc (test group) and (2)  . Immunoprecipitated proteins were collected by centrifugation at 5,000 g for 1 min at 4°C, washed three times with lysis buffer, The immunoprecipitates and the total cell lysates were boiled with 2× SDS sample buffer. The immunoprecipitates and total cell lysates were analyzed by IB with the indicated antibodies (Abs).

Statistical Analysis
The results of qRT-PCR data were reported as mean ± SEM of three independent experiments. Statistical analysis (unpaired t-test) was performed using GraphPad Prism 5 (GraphPad Software Inc.). A p < 0.05 was considered to be statistically significant.

Temporal Expression Pattern of CiPPP1R3G mRNA After the GCRV Infection
The distribution of CiPPP1R3G mRNA was detected in the spleen, liver, intestine, muscle and head-kidney tissues. Transcripts of CiPPP1R3G were expressed in all examined tissues, with the highest expression in the liver, and lowest in the intestine (Figure 2A). These data demonstrated that CiPPP1R3G is ubiquitously expressed in the tissues of grass carp. qRT-PCR was performed to investigate the expression of CiPPP1R3G in these tissues after the GCRV infection. The expression level of CiPPP1R3G reached a peak at 2 d in the muscle (16.15-fold, p < 0.001), at 3 d in the liver (5.88-fold, p < 0.001), head-kidney (236.01, p < 0.001) and intestine (205.73-fold, p < 0.01), and at 5 d in the spleen (9.25-fold, p < 0.001) ( Figure 2B).

The Expression of CiIRF3 Was Negatively Regulated by CiPPP1R3G
It is well known that IRF3 is a pivotal signalling molecule of the innate immune response. In the present study, to confirm the association of CiPPP1R3G and CiIRF3, we transfected expression plasmids of CiIRF3-Myc and CiPPP1R3G-Flag into HEK 293T cells and performed a Co-IP assay. Our results revealed that CiPPP1R3G interacted with CiIRF3 ( Figure 3A). In addition, compared with the control group, the expression of CiIRF3 was negatively regulated by the overexpression of CiPPP1R3G in a dose-dependent manner ( Figure 3B).  In this study, the activation of CiIFN1 caused by the CiIRF3 was investigated through a luciferase assay. As shown in Figure 4A, compared with the empty vector control group, CiIRF3 significantly upregulated the activation of the CiIFN1 promoter up to 34.27-fold. However, the activation of the CiIFN1 promoter was obviously reduced to 3.85-fold by the co-transfection of CiIRF3, CiPPP1R3G and the CiIFN1 promoter ( Figure 4A). Subsequently, we performed qRT-PCR assays to examine the expression of CiIFN1 after a challenge with GCRV in CIK cells. Consistently, overexpression of the CiPPP1R3G significantly attenuated the expression of CiIFN1 induced by the GCRV ( Figure 4B). Conversely, the effect of CiPPP1R3G knockdown on the expression of CiIFN1 was evaluated using siRNAs. As shown in Figure 4C, compared with cells transfected with control siRNAs (NC), the cells transfected with CiPPP1R3G-specific siRNAs (si-CiPP1#3) exhibited a significantly decreased level (50-60%) of CiPPP1R3G expression. The qRT-PCR analysis revealed that the knockdown of CiPPP1R3G increased the expression of CiIFN1 ( Figure 4D). Taken together, our results demonstrated that PPP1R3G can negatively mediate the activation of the IFN1 caused by the IRF3 in grass carp.

Subcellular Localization of CiPPP1R3G and CiIRF3
Subcellular localization of CiPPP1R3G was examined by transient transfection of the pAcGFP-N1-CiPPP1R3G plasmid into CIK cells.
The green fluorescent signal of pAcGFP-N1-CiPPP1R3G was distributed in the cytoplasm and nucleus of the CIK cells ( Figure  5A). Next, we co-transfected pAcGFP-N1-CiPPP1R3G with pM-RFP-CiIRF3. The red fluorescent signal of pM-RFP-CiIRF3 was mainly observed in the cytosol and almost overlapped with the green signal from CiPPP1R3G ( Figures 5B, C). Taken together, our data indicated that CiPPP1R3G was localized in the cytoplasm and nucleus, and associated with CiIRF3. morphological changes in the intestine after the GCRV-infection and high expression of CiPPPiR3G. As shown in Figure 6A, no pathological alterations were detected in the control group. In comparison with the control group, the intestine samples of GCRV-infected fish exhibited obvious lymphocyte infiltration into both LP (lamina propria) and IEL (intestinal epithelial layer), widened LP, narrowing of the interspace between the villi, and shortening of the height of MF (mucosal fold; p < 0.05; Figure 6B).

DISCUSSION
As an important phosphatase, PP1 participants in the regulation of a wide range of cellular events (4,5). Among them, it plays a pivotal role in the antiviral immune response. A previous study reported that PP1 can modulate the phosphorylation of the capsid of Venezuelan equine encephalitis virus (VEEV), and the inhibition of PP1 could slow down the viral replication in human cells  (U87MG astrocytoma cells, ATCC HTB-14) (27). In addition, it was revealed that PP1 can interact with the V proteins of measles virus and Nipah virus, thereby inhibiting dephosphorylation of MDA5 and eschewing the innate immune recognition of MDA5 (28).
As one of the regulatory subunits of the PP1, PPP1R3G has a major role in the regulation of postprandial glucose homeostasis during the fasting-feeding transition through its modulation of liver glycogenesis (6). PPP1R3G deletion accelerates the metabolic rate and reduces the glycogen level in adipose tissue in mice, indicating that PPP1R3G links glycogen and lipid metabolisms in vivo (29). In fish (silver carp Hypophthalmichthys molitrix), a study found that after a treatment with toxic Microcystis aeruginosa, PPP1R3G was down-regulated, indicating that it might play a major role in the detoxifying and antitoxic mechanisms of microcystin in fish (30).
In this study, a PPP1R3G protein was identified from grass carp and we found that it negatively regulates the function of IRF3 in the anti-GCRV immune responses. Temporal expression pattern revealed that CiPPP1R3G mRNA is highly expressed in several tissues; the highest in the liver, and relatively low in the intestine and head-kidney. As several previous studies reported, PPP1R3G plays an important role in the regulation of glycogenesis and glycogen metabolism in liver (6,29). Interestingly, after the GCRV stimulation, the mRNA level of CiPPP1R3G increased sharply in the intestine and head-kidney, indicating that these two tissues are the frontline of CiPPP1R3G to perform its function in the innate immune response against GCRV infection.
Fish intestine is the major site of immune responses in teleost fish (31)(32)(33)(34)(35)(36)(37). The immune cells that are essential for the gut immunization are plenteously present in the intestinal mucosa of teleost species (38). A previous study reported that the intestine is the main source of T cells in adult sea bass (Dicentrarchus labrax) (39). This tissue is also very important for anti-viral immune responses. For example, it was reported that IPNV (Infectious Pancreatic Necrosis Virus) infection could induce a differentiated epithelial immune response in the gut of Atlantic salmon (Salmo salar) (40). In the olive flounder (Paralichthys olivaceus), after the stimulation with VHSV (Viral Haemorrhagic Septicaemia Virus), a significant up-regulation of gene transcripts of IgT and its receptor pIgR (polymeric IgR) in the gut of the immunized fish (41). IgT is most abundant in mucosal compartments (42) and pIgR is strongly expressed in the gut enterocytes, where it recognizes the Ig molecules and transports them to the immune reaction site (31).
In the present study, we investigated the expression of PPP1R3G after the GCRV infection in grass carp. Interestingly, we found a significant up-regulation of the transcripts of PPP1R3G in the intestine compared with other tissues. An obvious histopathological alternation was detected in the intestine tissue of grass carp after GCRV infection. In conclusion, we found that the PPP1R3G play a negative role in the anti-GCRV immune responses. Further in vivo studies are needed to elucidate the molecular mechanisms of PPP1R3Gmediated signalling pathway in fish.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Research and Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.

AUTHOR CONTRIBUTIONS
HC conceived and designed the experiments. XH, BW, HF, MZ, and YL performed the experiments and analyzed the data. HC wrote the manuscript. All authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by National Natural Science Foundation of China (31972827).

ACKNOWLEDGMENTS
We thank Prof. Jianguo Su (Huazhong Agriculture University, China) for providing the plasmid of grass carp IFN1 (CiIFN1). We thank Dr. Fang Zhou (Institute of Hydrobiology, Chinese Academy of Sciences) for assistance with confocal microscopy analysis.