Cyprinid Herpesvirus 2 miR-C12 Attenuates Virus-Mediated Apoptosis and Promotes Virus Propagation by Targeting Caspase 8

DNA viruses, most notably members of the herpesvirus family, generally encode miRNAs to mediate both virus and host genes expression. We previously demonstrated that Cyprinid herpesvirus 2 (CyHV-2) encodes 17 miRNAs that are involved in innate immune signaling pathways. In this study, the function of CyHV-2-encoded miRNA was further investigated in GiCF cells. We found that miR-C4 promoted CyHV-2-induced apoptosis, while miR-C12 decreased CyHV-2-induced apoptosis. miR-C12 targeted to 3′ UTR sequence of caspase 8 and suppressed the expression of caspase 8. Besides, the silencing of caspase 8 by specific siRNA led to the attenuation of CyHV-2-induced apoptosis. Furthermore, caspase 8 was downregulated in cells transfected with miR-C12 during CyHV-2 infection. Overexpression of miR-C12 significantly suppressed CyHV-2-induced apoptosis, while silencing of miR-C12 promoted CyHV-2-induced apoptosis. Finally, inhibition of miR-C12 resulted in suppression of CyHV-2 propagation, overexpression of miR-C12, and CASP8-siRNA-1 facilitated CyHV-2 propagation. Taken together, our results demonstrated that CyHV-2-encoded miR-C12 to suppress virus-induced apoptosis and promoted virus replication by targeting caspase 8.

Apoptosis, or programmed cell death, is an energydependent process of cell suicide (Huang et al., 2014). Some studies reported that many different herpesviruses regulate apoptosis in infected cells (Miest et al., 2014;Scrochi et al., 2017;You et al., 2017). In our previous study, a novel cell line (Carassius auratus gibelio caudal fin cell line, GiCF) permission to CyHV-2 replication was established, and we demonstrated that CyHV-2 infection induces GiCF cell apoptosis (Lu et al., 2018a). MicroRNAs (miRNAs), are a class of small non-coding RNAs involved in post-transcriptional regulation of target gene expression in various biological processes (Ambros, 2004). A number of herpesviruses, including CyHV-2 and CyHV-3, encode miRNAs in infected cells. The regulatory effects of miRNAs involved in viral infection include apoptosis, oncogenic transformation, and modulation of viral life cycle (Donohoe et al., 2015;Bhela and Rouse, 2017;Lu et al., 2017). Viral miRNAs play an important role in the regulation of cell apoptosis, mostly by decreasing pro-apoptotic or anti-apoptotic genes expression (Pfeffer et al., 2004). For example, BCL2associated X (BAX) was demonstrated to be a target of viral miRNA ebv-miR-BART16 (Marquitz et al., 2011); BCL2associated death promoter (BAD) and the pro-apoptotic protein caspase 3 are targets of the ebv-miR-BART20-5p, ebv-miR-BART16 or ebv-miR-BART1-3p, respectively (Vereide et al., 2014). Our previous studies have identified 17 viral miRNAs from CyHV-2-infected crucian carp kidney, and have shown that the host genes PIN1, IRF3, and RBMX are the targets of CyHV-2-encoded miR-C4 . Recently, it was reported that host miRNAs are involved in CyHV-2 infection and participate in the regulation of apoptosis and immune-related genes (Lu et al., 2018b). However, whether CyHV-2-encoded miRNA has a regulatory effect on CyHV-2-induced apoptosis is still unknown.
Here, we report that miR-C12 encoded by CyHV-2 suppresses virus-induced apoptosis and promotes virus replication by targeting caspase 8. We show that overexpression of miR-C12 reduces the expression of caspase 8 and inhibits CyHV-2-induced apoptosis. In contrast, inhibition of miR-C12 promotes CyHV-2-induced apoptosis. We also demonstrate that downregulation of caspase 8 results in exacerbated CyHV-2 replication, while upregulation of caspase 8 results in CyHV-2 replication suppression. These results reveal that miR-C12 regulates CyHV-2-induced apoptosis through caspase 8.

Flow Cytometry Assay
Detection of cell apoptosis was conducted as previously described (Lu et al., 2018a). Briefly, cells were digested by 0.25% trypsin and stained for 20 min in the dark at room temperature with the Muse Annexin V and Dead Cell Reagent (Merck Millipore, USA). The stained cells was analyzed by Muse Cell Analyzer (Merck Millipore, USA), at least 10,000 events were collected for the cell gate.

miRNA Mimics and Inhibitors
All the miRNA mimics (dsRNA oligonucleotides) and miRNA inhibitors were commercially synthesized by Shanghai GenePharma (Shanghai, China) and the sequences were in Table 1. All the miRNA mimics, miR-NC, miR-C12 inhibitor, and inhibitor NC were transfected using RNAiMAX reagent (Invitrogen, USA).

RNA Interference
siRNAs were commercially synthesized by Shanghai GenePharma (Shanghai, China), and the siRNAs sequences were listed in Table 1. All the siRNAs were transfected into GiCF cells with 50 nM of each oligonucleotides using RNAiMAX reagent.

Plasmids
The 3′ UTRs of caspase 8 were amplified from GiCF cDNA, digested with Smal and Xhol, and ligated into pGL3-Basic Dual-Luciferase Vector (Promega, USA). Point mutation was conducted by the Fast Site-Directed Mutagenesis Kit (Tiangen, China) and the PCR primers were listed in Table 1. All the enzymes used for plasmid construction were produced by Takara (Dalian, China). The constructed plasmid sequences were verified by Sangon (Shanghai, China). Co-transfections with small RNA and pGL3 plasmids were performed with Lipofectamine 3000 (Invitrogen, USA).

Luciferase Assays
Hela cells transfected with pGL3 reporter plasmids and miR-C12 mimics, miR-NC, miR-C12 inhibitor, or inhibitor NC were analyzed post-transfection. The luciferase assays were conducted as previously described . The Dual-Glo luciferase assay kit (Promega, USA) was used to detect the quantity of firefly and Renilla luciferase by GloMax-Multi Detection System (Promega, USA). The relative luciferase activity was normalized to the levels in the renilla luciferase controls.

Western Blot Analysis
The cell extracts were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.8). Proteins were separated Frontiers in Microbiology | www.frontiersin.org 3 December 2019 | Volume 10 | Article 2923 by 10% SDS-PAGE, followed by transfer to PVDF membranes (GE Healthcare). Membrane was incubated overnight with primary antibody. After that, the membrane was incubated with secondary antibody conjugated with alkaline phosphatase (1:5,000) (Abmart, China). Finally, the membrane was conducted by ECL Western blot analysis kit (Thermo Fisher, USA). The optical density of the bands was quantified using TanonImage software (Tanon, China), caspase 8, or ORF72 protein expression was normalized to that of β-actin, representative blots of three independent experiments are shown. Caspase 8 polyclonal antibody was custom-made in GL Biochem Ltd. (Shanghai, China) by Peptides N2 (DHQKLHEIDEDLTST) and C7(RGHKQMPEPRYTLTKK). β-actin monoclonal antibody was supplied by Abmart (Wuhan, China), monoclonal antibody of ORF72 was produced in previous studies .

Real-Time Quantitative PCR
For mRNA quantification, total RNA was extracted using the TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instructions. Total RNA was reverse transcribed using the PrimeScript RT Master Mix (Takara, Japan). qRT-PCR was performed using the SYBR Premix Ex Taq (Takara), β-actin was used as an internal standard, the primers were listed in Table 1. At least, three independent biological replicates were used for each gene. All reactions were performed in triplicate on the CFX96 Real-time PCR Detection System (Bio-Rad, USA). Expression of the different genes was analyzed by 2 −ΔΔCT method.
The miRNA quantification was analyzed by Stem-loop qRT-PCR as described previously (Chen et al., 2005). The Hairpin-it™ MicroRNAs Quantitation PCR Kit custom-made in GenePharma was performed based on vendor protocols. Briefly, total RNA was first extracted using TRIzol reagent (Invitrogen, USA). For qRT-PCR, 1 μg of total RNA was reverse transcribed, and qPCR amplification was conducted in a 20 μl reaction. Synthetic pure miRNA was used as the standard. qPCR data normalized to total RNA was used to determine the miRNA copy number per 1 μg of total RNA. All the samples were carried out in triplicate and the data are represented as the mean ± SD.

Caspase 3/7 Activity
Caspase 3/7 activity of GiCF cells was conducted by caspase-Glo 3/7 assay kit (Promega, USA). In brief, GiCF cells were collected and washed with PBS, mixed the cells with Caspase-Glo 3/7 reagent, and incubated for 1 h at room temperature in the dark. Subsequently, the mixture was measured using a SPECTRA MAX 190 (Molecular Devices, USA).

Detection of CyHV-2 Copies
The detection of CyHV-2 copies was performed as described previously . Total DNA was extracted using a Tissue Genomic DNA Isolation Kit (Tiangen, China), 1 μl of genomic DNA was used for qPCR analysis. The qPCR conditions were as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 55°C for 30 s, and 95°C for 10 s, 65°C for 5 s, 95°C for 5 s. Real-time assays were performed in a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA).

Name
Sense Antisense Oligonucleotides for siRNA

Statistical Analysis
All data were analyzed by one-way ANOVA to calculate the means and standard deviations for triplicate assays, and the data were conducted using Student's t-test and a p <0.05 or < 0.01 were considered statistically significant.

miR-C12 Targets the 3′ UTR of Caspase 8
Next, we determined the mechanism of miR-C4 and miR-C12 in CyHV-2-induced cell apoptosis. In our previous research, caspase 8 was predicted as one of the candidate target gene of miR-C12, and we did not find a target gene directly related to apoptosis in miR-C4 . Therefore, miR-C12 was the main research object of this study. The binding site of miR-C12 to caspase 8 was shown in Figure 2A. The prediction results suggest that miR-C12 may be involved in the regulation of cell apoptosis.
To verify the predicted results, a dual-luciferase reporter assay was performed in HeLa cells. As shown in Figure 2B, the relative luciferase activity following co-transfection with miR-C12 mimics compared to transfection with control miRNAs (miR-NC) was reduced by 53.26% at 48 h and 79.42% at 24 h posttransfection, with no significant change in the 12 h post-transfection group. However, no significant change in luciferase was observed in cells transfected with mutant-type constructs 24 h posttransfection ( Figure 2C). Besides, miR-C12 expression level was increased by 2.14-fold on 24 h and 1.96-fold on 48 h in miR-C12 mimics transfected HeLa cells compared to 12 h post-transfection. In addition, miR-C12 expression level was increased by 1.79-fold on 24 h and 1.68-fold on 48 h in miR-C12 mimics transfected GiCF ( Figure 2D). We next investigated the mechanism responsible for the suppression of caspase 8 by miR-C12 in GiCF cells. To this end, we transfected miR-C12 mimics or control miRNA into GiCF cells and measured the mRNA and protein levels of caspase 8 in 12, 24, and 48 h post-transfection. As shown in Figures 2E-G Figure 2D). These findings suggest that miR-C12 targets 3′ UTR of caspase 8 and may play a key role in cell apoptosis.

miR-C12 Downregulates Expression of Caspase 8
To evaluate the effect of miR-C12 on caspase 8 expression, expression of miR-C12 was quantified during CyHV-2 infection by qRT-PCR. The results reveal that miR-C12 was detected from 6 h post CyHV-2 infection, and miR-C12 expression increased with the time of CyHV-2 infection ( Figure 4A). Inhibiting miR-C12 with the miR-C12 inhibitor results in downregulation of miR-C12 expression in CyHV-2-infected GiCF cells (0.65-fold), over expression of miR-C12 mimics led to the increase of miR-C12 expression by 19.75-fold compared to the miRNA control group in CyHV-2-infected GiCF cells ( Figure 4B). Next, miR-C12 mimics and inhibitor were used to evaluate the impact of overexpression or inhibition of miR-C12 on the expression of caspase 8 during CyHV-2 infection. As expected, the results shown that miR-C12 overexpression led to inhibition of caspase 8 mRNA levels by 0.56-fold and 0.39-fold in caspase 8 protein, miR-C12 inhibitor increased caspase 8 mRNA (2.19-fold) and protein levels (1.36-fold) at 24 h post infection (Figures 4C-E). The above data suggest that miR-C12 downregulates expression of caspase 8 during CyHV-2 infection.
To prove this hypothesis, we investigated the effect of miR-C12 on CyHV-2-induced cell apoptosis. Here, apoptosis was evaluated under conditions of miR-C12 knock down or overexpression.
inhibitor treated cells were decreased by 97.3% compared to the control group (1.2-fold). miR-C12 mimics led to the increase of CyHV-2 copies by 41.69-fold compared to the miRNA control group. miR-C12 mimics led to the increase of CyHV-2 copies by 41.69-fold compared to the miRNA control group. CASP8-siRNA-1 led to the increase of CyHV-2 copies by 16.6-fold compared to the control group ( Figure 6A). Besides, as a major capsid protein of CyHV-2, the expression trend of ORF72 protein was consistent with CyHV-2 copies. miR-C12 mimics and CASP8-siRNA-1 promoted ORF72 protein level by 1.42-and 1.39-fold, miR-C12 inhibitor treat resulted in an decrease of ORF72 protein level (0.54-fold) (Figures 6B,C). These findings indicate that CyHV-2 encodes miR-C12 to facilitate its propagation.

Model of miR-C12 Function in CyHV-2 Infection
Taken together, proposed model of miR-C12 in CyHV-2 infection is shown in Figure 7. Our studies demonstrated that CyHV-2 encodes miR-C12 and attenuates CyHV-2-induced apoptosis by targeting caspase 8. Subsequently, the inhibition of CyHV-2-induced apoptosis led to the increase of CyHV-2 propagation.

DISCUSSION
The aim of this study was to investigate the role of CyHV-2-encoded miRNA in CyHV-2-induced apoptosis. In this study, we found that miR-C4 promotes CyHV-2-induced apoptosis, while miR-C12 decreases CyHV-2-induced apoptosis. We also found that miR-C12 is employed by CyHV-2 to facilitate viral replication. Above all, our results demonstrate that CyHV-2encoded miR-C12 suppress virus-induced apoptosis and promotes virus replication by targeting caspase 8. A number of herpesviruses, especially gamaherpesvirus, have evolved strategies to encode viral miRNAs during  infection, counter host immunity through miRNA (Guo et al., 2015). Many virus-encoded miRNAs target the host gene mRNAs or viral genes to mediate the gene regulation function (Kincaid and Sullivan, 2012). Here, we found that miR-C4 promotes CyHV-2-induced cell apoptosis, while miR-C12 decreases CyHV-2-induced apoptosis. To promote infection, virus-encoded miRNAs mediate virus infection by targeting to host functional genes. There are several reasons for the opposite regulation of miR-C4 and miR-C12 in CyHV-2induced cell apoptosis. Firstly, miRNA was found to have a profound effect on the expression pattern of several hundred mRNAs (Reddy, 2015). One miRNA may target and regulate multiple genes, and one gene can be regulated by multiple miRNA (Qian et al., 2017). For example, several miRNAs in EBV cluster 1 have been identified to show both pro-apoptotic and anti-apoptotic effects (Vereide et al., 2014). Secondly, circular RNAs (circRNAs) or long noncoding RNA (LncRNA) interacted with miRNA, can reduce miRNA regulatory effect on mRNAs (Reddy, 2015;Kulcheski et al., 2016). miR-C4 might regulate CyHV-2-induced apoptosis through these unknown circRNAs or LncRNAs. Further work is necessary to clarify the exact mechanism of CyHV-2 miRNAs in virus-induced apoptosis. Besides, due to the limit of sequence depth and the absence of crucian carp genomic information, genes used to predict miRNA targets were incomplete. miR-C4 might regulate CyHV-2-induced apoptosis through unknown genes. Viruses have evolved various strategies to repress apoptosis by regulating pro-apoptotic and anti-apoptotic factors. During its life cycle, α-herpesvirus utilizes various anti-apoptotic strategies to suppress programmed cell death (You et al., 2017). Viral miRNAs target both host and viral mRNAs, and regulate virus-induced apoptosis by targeting to pro-apoptotic or anti-apoptotic factors. By targeting host mRNAs, viral miRNAs can regulate genes involved in apoptosis, immune response, cell differentiation, cell cycle control, and intracellular trafficking (Piedade and Azevedopereira, 2016). In this study, caspase 8 was confirmed as a target of miR-C12, and miR-C12 suppresses CyHV-2-induced apoptosis by targeting caspase 8 in GiCF cells. As an initiator and apical activator caspase, caspase 8 plays a key role in extrinsic apoptosis (Tummers and Green, 2017). Several miRNAs mediate apoptosis by targeting caspase 8. For instance, host miR-134 negatively regulates the oligodendrocytes (OLs) apoptosis by targeting caspase 8 (Xiao et al., 2019). WSSVencoded WSSV-miR-N24 represses the apoptosis of shrimp hemocytes by targeting caspase 8 in shrimp hemocytes (Huang et al., 2014). In conclusion, to facilitate viral infection, CyHV-2-encoded miR-C12 to suppress virus-induced apoptosis by targeting to caspase 8.
Several reports have shown that virus-encoded miRNAs influence virus replication and propagation. Viral miRNAs are expressed during infection, suggesting that viral miRNA might participate in virus propagation and survival (Qian et al., 2017). For example, WSSV-miR-N24 inhibits cell apoptosis through increase in caspase 8 expression, leading to an increase in WSSV copy number (Huang et al., 2014). Furthermore, kshv-miR-K12-5 and kshv-miR-K12-9 target BCLAF1, and promote KSHV lytic replication (Bellare and Ganem, 2009). In contrast, host miRNA can inhibit the replication of hepatitis B virus by regulating the expression of a host gene that is beneficial to virus infection (Zhao et al., 2014). Notably, WSSV and KSHV encode miRNAs, suggesting that these viruses have evolved to utilize the expression of viral miRNA to promote their propagation. In this study, we found overexpression of miR-C12 mimics led to a significant increase of CyHV-2 copies, while suppressing miR-C12 with the miR-C12 inhibitor resulted in a decrease in CyHV-2 replication. Caspase 8 increased with CyHV-2 infection and viral miR-C12 decreased apoptosis; this indicated that the CyHV-2 infection increases the expression of caspase 8, and miR-C12 was serving to counteract this effect. The complexity of the miRNA-mRNA interaction was also observed in WSSV-and HIV-encoded miRNA (Klase et al., 2009;Huang et al., 2014). Collectively, our results demonstrate that miR-C12 is employed by CyHV-2 to regulate host caspase 8 expression to promote viral propagation through inhibition of cell apoptosis.
In summary, our results reveal that CyHV-2 miR-C12 is an important suppressor of CyHV-2-induced apoptosis by downregulating caspase 8 expression. Therefore, it seems that using miR-C12 can be an effective method for CyHV-2 to control distinct host genes and establish an ideal environment for viral propagation, or prevent CyHV-2-infected cell from increasing host immune responses.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/ supplementary material.

ETHICS STATEMENT
All animal work in this paper was conducted according to relevant national and international guidelines. All animal care and experimental procedures were approved by the Committee on Animal Care and Use and the Committee on the Ethics of Animal Experiments of Ningbo University.

AUTHOR CONTRIBUTIONS
JL, DX, and LL were responsible for the experimental design, analyses, and interpretation of the data. JL drafted the manuscript. JL and ZS performed the experiments. All authors read and approved the final version of the manuscript.