key: cord-335393-4buooi2d
authors: Xiang, Yangxi; Jia, Peng; Liu, Wei; Yi, Meisheng; Jia, Kuntong
title: Comparative transcriptome analysis reveals the role of p53 signalling pathway during red‐spotted grouper nervous necrosis virus infection in Lateolabrax japonicus brain cells
date: 2019-01-18
journal: J Fish Dis
DOI: 10.1111/jfd.12960
sha: 
doc_id: 335393
cord_uid: 4buooi2d

Nervous necrosis virus (NNV) is one of the fish pathogens that have caused mass mortalities of many marine and freshwater fishes in the world. To better comprehend the molecular immune mechanism of sea perch (Lateolabrax japonicus) against NNV infection, the comparative transcriptome analysis of red‐spotted grouper nervous necrosis virus (RGNNV)‐infected or mock‐infected L. japonicus brain (LJB) cells was performed via RNA sequencing technology. Here, 1,969 up‐regulated genes and 9,858 down‐regulated genes, which were widely implicated in immune response pathways, were identified. Furthermore, we confirmed that p53 signalling pathway was repressed at 48 hr post‐RGNNV infection, as indicated by up‐regulation of Mdm2 and down‐regulation of p53 and its downstream target genes, including Bax, Casp8 and CytC. Overexpression of L. japonicus p53 (Ljp53) significantly inhibited RGNNV replication and up‐regulated the expression of apoptosis‐related genes, whereas the down‐regulation caused by pifithrin‐α led to the opposite effect, suggesting Ljp53 might promote cell apoptosis to repress virus replication. Luciferase assay indicated that Ljp53 could enhance the promoter activities of zebrafish interferon (IFN)1, indicating that Ljp53 could exert its anti‐RGNNV activities by enforcing the type I IFN response. This study revealed the potential antiviral role of p53 during NNV infection.

HSP27 and RAVER1 , have been identified and characterized in sea perch. However, knowledge about the immunity system of sea perch, especially the immune response-related signalling pathways implicated in NNV infection, still remains incomplete. Systematic study of sea perch immune-related signalling pathway is necessary to provide the fundamental understanding of the interaction between sea perch and NNV.

Transcriptome analysis is a fast and cost-effective way to understand the underlying pathways and mechanisms of host against pathogen infection by evaluating immune responses of gene expression (Liu, Wang, Kwang, Yue, & Wong, 2016; Zhong et al., 2017) .

Many aquatic species have been sequenced to study pathogenic processes during virus infection, including mandarin , orange-spotted grouper (Huang et al., 2011) , Pacific white shrimp (Zeng et al., 2013) and rainbow trout (Aquilino, Castro, Fischer, & Tafalla, 2014) .

It was well known that the brain and retina were the main target organs of NNV (Poisa-Beiro et al., 2008) . Therefore, cells originated from sea perch brain should be a good material for studying sea perch-NNV interaction. In our previous study, a continuous cell line L. japonicus brain (LJB), derived from the brain of sea perch, was established and exhibited susceptibility to red-spotted grouper nervous necrosis virus (RGNNV) (Le, Li et al., 2017) . In this study, transcriptome sequencing libraries were constructed with RGNNV-infected or mock-infected LJB cells.

Based on the analysis of differentially expressed genes (DEGs), we found that p53 signalling pathway might be involved in the immune response against RGNNV, and experimentally revealed the role of L. japonicus p53 (Ljp53) in the regulation of the type I IFN response and cellular apoptosis during RGNNV infection.

This study provides insight into the immune response of sea perch against RGNNV infection and the important role of Ljp53 in inhibiting RGNNV infection.

Lateolabrax japonicus brain cells were grown and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% foetal bovine serum (FBS) (Gibco) at 28°C (Le, Li et al., 2017) .

HEK293T (human embryonic kidney 293) cells were maintained in DMEM supplemented with 10% FBS at 37°C in a 5% CO 2 incubator (Russell, Graham, Smiley, & Nairn, 1977) .

Red-spotted grouper nervous necrosis virus was isolated from diseased sea perch in Guangdong Province of China and kept in our laboratory . RGNNV was propagated in LJB cells at 28°C, and virus titres were detected by 50% tissue culture infective dose (TCID 50 ) (Jia, Zhang et al., 2015) .

Zhang at the Institute of Hydrobiology, Chinese Academy of Sciences (Zhang & Gui, 2012) .

Lateolabrax japonicus brain cells were infected with RGNNV (multiplicity of infection [MOI] = 5) at 28°C for 4 hr. Then, the medium containing RGNNV was discarded and the same volume of growth medium with 15% FBS was added. The control (mock-infected LJB cells) was treated with the same volume of medium. RGNNV-infected or mock-infected LJB cells were harvested for RNA isolation at 48 hr post-infection (hpi), respectively. Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The concentration of total RNA was determined using NanoDrop 2000 UV-Vis Spectrophotometer.

For cDNA library construction and sequencing, mRNA was first isolated from total RNA treated with DNase I using Magnetic Oligo (dT) Beads (Illumina) and was fragmented. Then, the double-stranded cDNA was synthesized with random hexamer primers and was further subjected to end-repair and adapter ligation using T4 DNA ligase.

The products of ligation reaction were purified on 2% agarose gel, and cDNA fragments (about 200 bp) were recovered. PCR was carried out to enrich the purified cDNA template. Finally, the cDNA library was constructed. After validating on an Agilent Technologies 2100 Bioanalyzer, the library was sequenced using Illumina HiSeq 4000 according to the manufacturer's instruction. All data sets have been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database PRJNA497762.

After sequencing, we carried out a stringent filtering process of raw sequencing reads as described previously (Gui et al., 2013) . The raw reads were cleaned by removing adapter sequences, non-coding RNA, low-quality sequences (reads with ambiguous bases "N" and the ratio of "N" > 10%) and reads with average length <20 bases.

De novo transcriptome assembly was performed by Trinity program (v2.0.6) as described elsewhere (full-length transcriptome assembly from RNA-seq data without a reference genome). The function of unigenes was annotated depending upon following databases: NR (NCBI non-redundant protein sequences), NT (NCBI nucleotide sequences), GO (Gene Ontology), COG (Clusters of Orthologous

Genomes) and Swiss-Prot (a manually annotated and reviewed protein sequence database).

For differential gene expression analysis, reads per kilobase of exon model per million mapped reads value was used to normalize the gene expression levels (Poisa-Beiro et al., 2008) . Statistical comparison between two different groups was conducted using a web tool DESeq (http://www-huber.embl.de/users/anders/DESeq) (Lu et al., 2017) . False discovery rate (FDR) <0.05 was used as the threshold of p-value in multiple tests to judge the significance of gene expression difference. Genes were considered differentially expressed in a given library when the p-value <0.05, and a greater than twofold change (absolute value of log2 ratio >1) in expression across libraries was observed. GO and KEGG pathway enrichment of differential unigenes were analysed. KO analysis of differential expression unigenes in metabolic pathways was conducted on website (http:// www.genome.jp/kegg/tool/map_pathway2.html).

Red-spotted grouper nervous necrosis virus infection and sample collection were performed as described above. Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA by PrimeScript™ First Strand cDNA Synthesis Kit (Takara) according to the manufacturer's instructions. The relative expression levels of Mdm2, Mdm4, p53, Bax, Casp8 and Casp9 were analysed by quantitative real-time PCR (qRT-PCR). qRT-PCR was performed as described previously (Le, Li et al., 2017) . Primers for qRT-PCR are listed in Supporting Information Table S1 . Expression levels of target genes were normalized with β-actin of sea perch by the 2 −ΔΔCT methods. Data from each sample were shown as mean ± SD from three independent experiments in triplicates.

To investigate the effects of Ljp53 overexpression on RGNNV replication, encoding region of Ljp53 was amplified by PCR using gene-specific primers and sub-cloned into pcDNA 3.1 (+) vectors (Invitrogen) to generate pcDNA-Ljp53, which was confirmed through DNA sequencing analysis (Supporting Information Table   S1 ). LJB cells in six-well plates at 70%-80% confluence were transfected with pcDNA-Ljp53 or pcDNA 3.1(+) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instruction. At 24 hr post-transfection, cells were infected with RGNNV (MOI = 1) and harvested at 48 hpi for total RNA isolation. The expression of RNA-dependent RNA polymerase (RDRP) was detected by qRT-PCR as described above. Primers for qRT-PCR are listed in Supporting Information Table S1 .

To evaluate the effect of down-regulation of Ljp53 on RGNNV replication, LJB cells in six-well plates at 70%-80% confluence were treated with p53 inhibitor pifithrin-α (MedChemExpress) at concentration of 20 μM or DMSO (0.1%) for 2 hr, and then, the treated cells were infected with RGNNV (MOI = 1) in the presence of pifithrin-α or DMSO. Cells were harvested for qRT-PCR at 48 hpi. qRT-PCR was performed as described above. Primers for qRT-PCR are listed in Supporting Information Table S1 .

Luciferase activity assay was carried out as described previously . In brief, the plasmids pRL-TK (Promega), pcDNA-Ljp53 or pcDNA 3.1(+), and DrIFN1-pro-luc were co-transfected into HEK293T cells using Lipofectamine 3000. At 48 hr post-transfection, cells were harvested with passive lysis buffer and subjected to luciferase activity measurement using GloMax 20/20 Luminometer (Promega).

Data were expressed as mean ± SD from three independent experiments performed in triplicates.

To evaluate the effect of Ljp53 on apoptosis-related genes during RGNNV infection, the encoding region of Ljp53 was sub-cloned into pEGFP-N3 vectors (Invitrogen) to generate pEGFP-Ljp53, which was confirmed through DNA sequencing analysis (Supporting Information Table S1 ). LJB cells were transfected with pEGFP-Ljp53 or pEGFP-N3 or treated with pifithrin-α as above. Finally, cells were harvested for RNA isolation at 48 hpi. The relative expression levels of Casp3, Casp9 and CytC were analysed by qRT-PCR. Primers for qRT-PCR are listed in Supporting Information Table S1.

All statistics were calculated using SPSS version 19. Differences between control and treatment groups were assessed by one-way ANOVA. p < 0.05 (*) and p < 0.01 (**) were, respectively, considered to be statistically significant and very significant.

To understand the gene expression patterns of LJB cells in response to RGNNV infection, RNA-seq libraries were generated from RGNNV-infected and mock-infected LJB cells. A total of 4.751 × 10 7 and 4.525 × 10 7 raw reads were produced in mock-infected and RGNNV-infected samples, respectively. After filtration, 4.51 × 10 7 and 4.485 × 10 7 clean reads obtained from the mock-infected sample (control group) and RGNNV-infected group were used for de novo assembly (Table 1 ). In control group, transcriptome assembly unigenes was the most highly represented group (Figure 2 ). COG classification indicated that 9,001 unigenes were assigned to 25 different COG categories. The cluster for "General function prediction only" represented the largest group (3,237 unigenes), followed by "replication, recombination and repair" (1,426 unigenes) and "transcription" (1,375 unigenes) (Figure 3) . A total of 18,029 unigenes were assigned to 301 KEGG pathways, covering six main categories, include cellular processes, environmental information processing, genetic information processing, human diseases, metabolism and organismal systems. Metabolic pathway (1,883 unigenes) was the most abundant KEGG pathway, followed by "pathway in cancer" (844 unigenes) and "focal adhesion" (678 unigenes) (Supporting Information Table S2 ).

To identify DEGs, the unigenes between LJB-Control and LJB-NNV groups were compared. A large number of genes were found to be differentially expressed in LJB cells post-RGNNV infection, including 1,969 up-regulated genes (DUGs) and 9,858 down-regulated genes (DDGs) (FDR ≤ 0.001 and log2 ratio ≥ 1) (Supporting Information Table S3 ). Of these genes, many well-known immune-related genes were strongly inhibited after RGNNV infec- 

Comparative transcriptome analysis showed that 79 DEGs involved in p53 signalling pathway were regulated in RGNNV-infected LJB cells compared to control cells (Supporting Information Figure S1 ). These results suggested that the expression profiling of DEGs determined by RNA-seq was reliable. These results indicated that p53 signalling pathway was not only involved in RGNNV infection but also inhibited by RGNNV at 48 hpi. 

Given the important role of p53 in innate antiviral immunity, in the pre- Many works have demonstrated that p53 contributed to the innate antiviral response by enhancing type I IFN-dependent antiviral activity (Ding et al., 2018; Muñoz-Fontela et al., 2008) . To further reveal the potential antiviral mechanism of Ljp53, the luciferase reporter assay was performed. Our results indicated that zebrafish IFN1 promoter was activated by Ljp53 overexpression (Figure 5c ), indicating that Ljp53 might exert its anti-RGNNV activity partially by activating the IFN signalling pathway.

To validate the antiviral role of Ljp53 was associated with its proapoptotic activity in RGNNV infection, the mRNA levels of Casp3,

Casp9 and CytC were measured in Ljp53 overexpressing LJB cells.

Our results showed that overexpression of Ljp53 up-regulated the transcription of pro-apoptotic Casp3, Casp9 and CytC at 48 hpi, whereas the suppression of Ljp53 caused by pifithrin-α led to the opposite effect ( Figure 6 ). Thus, our results suggested that the apoptosis induced by RGNNV infection was dependent to some extent on Ljp53, and Ljp53 might be involved in promoting apoptosis in LJB cells.

Sea perch is a commercially important marine fish widely cultured in Asia. However, the high mortality in a wide range of larvae and juveniles caused by NNV is an intensive threat. Nevertheless, there is limited transcriptomic information available in relation to the molecular immune mechanism of sea perch against NNV infection. In this study, transcriptome sequencing libraries were generated from RGNNV-infected or mock-infected LJB cells. In total, 4.51 × 10 7 and 4.485 × 10 7 clean reads were generated from the control group and RGNNV-infected group, and 39,954 unigenes were annotated by six databases. And the number of unigenes was slightly different from previous reports in which the fish were infected with different pathogens (Lu et al., 2017; Zhao et al., 2016) . to escape the antiviral response, including counteracting the activity of ISG15 (Guerra, Cáceres, Knobeloch, Horak, & Esteban, 2008; Yuan & Krug, 2001) . Several studies have addressed NNV persistent infections in many fish, indicating NNV has developed some mechanisms to antagonize host innate antiviral responses (Lu et al., 2016; Zhang et al., 2017) . We speculated that RGNNV might counteract the antiviral activity of ISG15 to escape the host antiviral response in LJB cells. Zinc finger protein 395 (ZNF395) is one of the most downregulated genes (decreased by 18.11 times) in this study. ZNF395, as a novel hypoxia-inducible transcription factor, contributes to the maximal stimulation of a subset of ISGs, such as ISG15, IFIT1/ISG56, IFI44, CXCL10 and CXCL11 (Schroeder, Herwartz, Jordanovski, & Steger, 2017) . In this study, in line with the down-regulation of ISG15, the expression of ZNF395 was also decreased. Given these results, it is possible that the declining level of ZNF395 may impair host antiviral responses by reducing the expression of ISGs involved in the innate immune response during RGNNV infection. It was known that ZNF395-mediated activation of ISGs was depended on IKK signalling (Jordanovski et al., 2013) . In present work, the expression levels of IKKα and IKKβ, the catalytic subunits of IKK, were also repressed post-RGNNV infection. Further experiments would be necessary to determine the association of ISG15, ZNF395 and IKK signalling in sea perch. In addition, among these DEGs, HSPB1, also named HSP27, was down-regulated, similar to our previous study . HSPB1 has been reported to be widely involved in pathophysiology of oxidative stress and apoptosis. Overexpression of HSPB1 protected cells from oxidative damage and apoptosis triggered by Cd exposure (Munōz-Fontela, 2005) . In our previous study, overexpression of LjHSP27 inhibited RGNNV-induced apoptosis, supporting the vital role of HSP27 as an anti-apoptosis protein . It has been known that many DNA and RNA viruses can trigger oxidative stress and induce host cell death in infected cells (Casavant et al., 2006; Yuan et al., 2015) . The role of LjHSP27 against oxidative stress still needs further research.

Published literature about the immune-related signalling pathways involved in RGNNV infection was mainly focused either on the apoptosis pathway or on the innate immune pathways Wang, Rajanbabu, & Chen, 2015 Tumour suppressor p53, primarily famous for its vital role in protecting against cancer development, has been proven to regulate various biological process, such as cell cycle arrest, cellular metabolism, cellular apoptosis and innate antiviral immunity (Liu, Zhang, Hu, & Feng, 2015) . Several studies indicated that p53 functioned as a key player in innate antiviral immunity by both enforcing the type I IFN response and inducing apoptosis in virus-infected cells (Rivas, Aaronson, & Munoz-Fontela, 2010) . Here, the expression of RDRP was significant reduced in Ljp53 overexpressing cells post-RGNNV infection, whereas pifithrin-α caused opposite results. Furthermore, the results of luciferase reporter assay showed that zebrafish IFN1 promoter was activated by Ljp53 overexpression. Thus, we speculated that Ljp53 inhibited RGNNV replication by activating the IFN signalling pathway.

It was known that the role of p53 in the control of virus infection was also associated with its ability to activate apoptosis during virus infection, which inhibited virus replication (Rivas et al., 2010) . Several studies have reported that p53 can induce apoptosis through activating death receptor pathway or mitochondrial pathway (Benchimol, 2001; Moll & Zaika, 2001) . Our previous study demonstrated that RGNNV infection caused apoptosis at 6 and 24 hpi in LJB cells . Meanwhile, our results indicated that three apoptosis-related genes, including Casp3, Casp9 and CytC, were notably enhanced as the ectopic expression of Ljp53 in vitro, whereas pifithrin-α caused the opposite effect.

In addition, significantly higher level of caspase 3 activities was observed in Ljp53 overexpressing cells post-RGNNV infection.

All these results indicated the pro-apoptotic function of Ljp53 (Supporting Information Figure S2 ). It has been known that p53dependent apoptosis could be used by host as a useful mechanism to repress virus infection (Mun̄oz-Fontela et al., 2005; Turpin et al., 2005) . On the other hand, many viruses have also developed strategies to manipulate host p53 signalling pathways to increase virus replication (Casavant et al., 2006; Royds et al., 2006) . For example, coronavirus inhibited p53-mediated host apoptosis to ensure viral growth in infected cells (Yuan et al., 2015) . In our study, RGNNV infection reduced the expression of Ljp53 at 48 hpi, which at least partially inhibited the host cell apoptosis. We speculated that RGNNV might repress the p53-dependent apoptosis to promote virus replication in RGNNV-infected LJB cells and facilitate transmission of newly formed viral particles to other cells.

In this study, transcriptome sequencing libraries were constructed with LJB cells mock-infected or infected with RGNNV at 48 hpi. A large number of DEGs in response to RGNNV infection were identified. The DEGs involved in p53 signalling pathway were further investigated. Our results demonstrated that Ljp53 played an essential role in inhibiting RGNNV replication. Mechanistically, we confirmed that the antiviral role of Ljp53 was involved in its activity in mediating the IFN response and its pro-apoptotic activity.

This work was supported by the National Natural Science 

The authors declare no competing financial interests.

Meisheng Yi https://orcid.org/0000-0003-1794-2734

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