MD-2 Homologue Recognizes the White Spot Syndrome Virus Lipid Component and Induces Antiviral Molecule Expression in Shrimp

Jie Gao, Jin-Xing Wang and Xian-Wei Wang
J Immunol September 1, 2019, 203 (5) 1131-1141; DOI: https://doi.org/10.4049/jimmunol.1900268

Key Points

  • Shrimp MD-2 homologue ML1 recognizes a lipid component of white spot syndrome virus.

  • Shrimp ML1 activates NF-κB signaling.

  • ML1 induces the expression of Vago, which is a functional analogue of IFN.

Abstract

The myeloid differentiation factor 2 (MD-2)–related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann–Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host–virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus. One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD–ML1–Dorsal–Vago signaling provided new insights into invertebrate antiviral immunity.

Introduction

Myeloid differentiation factor 2 (MD-2)–related lipid-recognition (ML) family proteins are characterized by the presence of an ML domain. Comprising ∼150 residues, the ML domain shows an overall structure of two antiparallel β sheets that are built from multiple β strands. The two sheets enclose a cavity to accommodate lipids or lipid-like molecules. The hydrophobic residues in the binding pocket are mainly responsible for interacting with the side chains of lipids. A high degree of sequence variation in the ligand-binding site, especially the hydrophobic residues, allows the ML domain to recognize a variety of lipids (1). Through interacting with specific lipids originated from nonself or self via the ML domain, the ML family is mainly involved in antibacterial immunity and lipid metabolism, with MD-2 and Niemann–Pick disease type C2 (NPC2), respectively, as representatives for the two typical roles.

MD-2 was identified as an accessory receptor accompanying TLR4 in LPS sensing (2). MD-2 is coexpressed with TLR4, and the TLR4/MD-2 heterodimer forms before LPS binding (3). The hydrophobic cavity of MD-2 provides a site for five of six acyl chains of LPS. The remaining exposed acyl chain of LPS interacts with the conserved hydrophobic phenylalanines located in the C terminus of the extracellular domain of a second TLR4, leading to dimerization of two TLR4–MD-2 complexes (4, 5). The close proximity of the TLR4 intracellular domains results in the recruitment of downstream adaptors, the initiation of an immune signaling cascade, and finally, the expression of inflammatory cytokines (6). MD-1, an ortholog of MD-2 sharing ∼20% sequence homology with MD-2, is also involved in LPS signaling together with radioprotective 105, which shares 30% sequence identity with TLR4 (7, 8). The radioprotective 105/MD-1 complex promotes LPS-induced cell growth and Ab production in B cells and suppresses LPS-induced immune signaling in dendritic cells and macrophages by interacting with the TLR4/MD-2 complex (9, 10).

NPC2 was named for its involvement in the NPC2, which is a neurodegenerative lysosomal lipid storage disorder (11). Mutation of the NPC2 gene would lead to the impairment of egress of cholesterol from lysosomes. The NPC2 protein is secreted, recaptured from extracellular sites, and transported into lysosomes. By binding and transferring cholesterol in late endosomes or lysosomes to NPC1, another important participator in cholesterol trafficking, NPC2 plays an important role in regulating intracellular cholesterol homeostasis (12). The structural basis of NPC2-cholesterol binding has been revealed. Unliganded bovine NPC2 adopts an overall Ig-like β-sandwich structure, similar to other ML proteins. The hydrophobic interior core of bovine NPC2 provides a site to accommodate cholesterol (13). Similar to NPC2, GM2 activator (GM2A) is also a small protein involved in lipid metabolism (14). Through its lipid-binding ability, GM2A functions as a cofactor for β-hexosaminidase A, which converts ganglioside GM2 to GM3. Deficiency or functional impairment of GM2A leads to GM2 accumulation and neuronal disease (15).

In addition to LPS signaling and lipid metabolism, studies have suggested the importance of the ML family in host–virus interaction. For example, mouse mammary tumor virus enhances its infectivity by acquiring host LPS binding factors, including CD14, TLR4, and MD-2, to exploit LPS from commensal bacteria (16). Human MD-2 interacts with the respiratory syncytial virus (RSV) fusion (F) protein to activate TLR4–NF-κB–mediated cytokine expression (17). Loss of host NPC2 greatly increased the cholesterol level in late endosomes/lysosomes and enhanced HIV infectivity (18). The involvement of ML proteins in host–virus interaction was also observed in invertebrates. Silencing of a dengue virus–inducible ML protein in Aedes aegypti, AaegML33, resulted in a significantly lower dengue virus titer in the mosquito midgut. Further study showed that AaegML33 likely facilitated viral infection as a potential negative regulator for the JAK/STAT pathway and the immune deficiency pathway (19, 20). In a study to identify the proteins responsible for white spot syndrome virus (WSSV) resistance in Pacific white shrimp (Litopenaeus vannamei), an ML family member was found to be more abundant in virus-resistant shrimp than in virus-susceptible shrimp, suggesting its possible role in WSSV resistance (21). We also observed the frequent appearance of ML family proteins after WSSV infection in kuruma shrimp (Marsupenaeus japonicus) when attempting to identify virus-inducible genes using transcriptomic analysis (J. Gao, J.X. Wang, and X.W. Wang, unpublished observations).

All above information indicated that the ML family plays important roles in the host–virus interaction. However, the specific mechanism remains largely elusive. In the current study, we investigated the participation of the kuruma shrimp ML family in WSSV infection. Specifically, the function and mechanism of an MD-2 homologue was studied. The finding that the shrimp MD-2 homologue recognizes a WSSV envelope lipid component and regulates the expression of antiviral molecules provides new insights into the significance of the ML family in host–virus interaction.

Materials and Methods

Animal cultivation, virus challenge, and sample preparation

Healthy kuruma shrimp (M. japonicus; 3–5 g) were purchased from a market in Jinan, Shandong, China and cultivated in air-pumped artificial seawater (25°C) for at least 1 wk before the experiments. The WSSV strain used in this study was collected from infected red swamp crayfish (Procambarus clarkii), and the original inoculum was gifted from the East China Sea Fisheries Research Institute (Shanghai, China). Shrimp were artificially infected via i.m. injection with the original inoculum, and moribund shrimp were collected and stored at −80°C before use for WSSV propagation. To prepare successive inoculums, shrimp gills (1 g) were homogenized in 10 ml of PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 [pH 7.4]). The homogenate was frozen and thawed twice and then centrifuged at 3000 rpm × g for 10 min at 4°C. The supernatant was filtered through a 0.45-μm filter. The filtrate was used as the WSSV inoculum. Viral DNA was extracted from 100 μl of the filtrate using MagExtractor Genome (TOYOBO, Shanghai, China) to determine the viral titer according to a previously described method (22). The rest of the filtrate was stored at −80°C and diluted to the appropriate titer with PBS before use. For WSSV challenge, each shrimp was injected with WSSV inoculum at a dose of 5 × 105 virus copies. PBS was injected as the control. At different times postinfection, shrimp hemolymph was collected into cold anticoagulant (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, and 10 mM HEPES [pH 7.45]) and centrifuged at 800 rpm × g for 7 min at 4°C to obtain the hemocytes pellet. Other tissues were collected simultaneously. Each sample originated from at least five shrimp. Total RNA was extracted using TRIzol (Invitrogen, Waltham, MA) from ∼100 mg of tissue or 2 × 107 cells, and the first stranded cDNA was synthesized using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO) according to the manufacturers’ instructions.

Expression profile analysis

Semiquantitative RT-PCR was performed to study the tissue distribution of MjML mRNAs using the gene-specific primers listed in Table I, following a conventional procedure: 94°C for 3 min; 30 cycles of 94°C for 15 s, 54°C for 30 s and 72°C for 30 s; and a final 72°C for 10 min, using the EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China). PCR products were analyzed using 1.5% agarose gel electrophoresis. Quantitative real-time RT-PCR (qRT-PCR) was performed to determine the expression profiles of MjML mRNAs upon WSSV challenge using the iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) and the CFX96 Real-Time System (Bio-Rad Laboratories), using the same primers used for RT-PCR. The PCR procedure was as follows: 94°C for 5 min, followed by 40 cycles of 94°C for 10 s and 60°C for 1 min, and a final melting from 65°C to 95°C. PCR data were analyzed using the 2−△△Ct method. β-Actin was used as the internal reference for both semiquantitative RT-PCR and qRT-PCR. The expression level was normalized to the control group at each time point. Three independent experiments were performed, and the results represented the mean ± SD.

Bioinformatics analysis

The human MD-2 sequence was used as a template to perform local basic local alignment search tool searching using BioEdit software to find possible homologues from the transcriptome data obtained from our previous transcriptome sequencing of healthy shrimp, which was performed by BGI Group (Shenzhen, China). The domain architecture was predicted using SMART (http://smart.embl-heidelberg.de/). A neighbor-joining phylogenetic tree was built using MEGA 6.0 with 1000 bootstraps. Multiple alignment of protein sequences was performed using the online tool ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/). The secondary structure was predicted using the online tool PredictProtein (https://www.predictprotein.org/). The promoter sequence was analyzed using Promoter Scan online (https://www-bimas.cit.nih.gov/molbio/proscan/).

RNA interference

A partial DNA fragment was amplified using primers linked to a T7 promoter (Table I). The products were used as templates to synthesize dsRNA using an in vitro T7 Transcription Kit (Takara Bio, Dalian, China). dsGFP was synthesized as a control. The oligonucleotides containing the T7 promoter and the small interfering RNA (siRNA) sequence (Table I) were commercially synthesized and used as templates to synthesize siRNA, using the in vitro T7 Transcription Kit (for siRNA synthesis) (Takara Bio). For each gene, two specific siRNAs were synthesized and mixed for use. The siRNA specific for GFP sequence was synthesized as a control. Specific dsRNAs or siRNAs (30 μg) were injected into the shrimp hemocoel at the abdominal segment, and the control group was injected with an equal amount of control dsRNA or siRNA. The RNA interference (RNAi) efficiency was determined using qRT-PCR at 48 h after dsRNA injection or 24 h after siRNA injection.

To test the function of MjMLs in virus infection, WSSV infection (5 × 105 copies per shrimp) was performed at 48 h after dsRNA or 24 h after siRNA injection. The expression level of WSSV VP28 was then analyzed using qRT-PCR with the primers listed in Table I. The experiments were performed independently three times.

Generation of recombinant protein

The sequence encoding the MjML1 mature peptide was amplified using the specific primers listed in Table I and ligated into the pET32a(+) plasmid. The recombinant vector was transformed into Escherichia coli Rosetta (DE3) strain for expression under induction with 0.5 mM isopropyl-β-d-thiogalactopyranoside. Inclusion bodies were extracted, washed, and dissolved in buffer (0.1 mM Tris-HCl [pH 8], 10 mM DDT, and 8 M urea) and renatured by dialysis in PBS with 5% glycerol. Recombinant proteins were then purified using affinity chromatography with ProteinIso Ni-NTA Resin (TransGen Biotech). Endotoxins were removed by thorough washing with cold 0.1% Triton X-114 before the final elution of the protein from the column (23). Purified proteins were then dialyzed in PBS and stored at −80°C before use. A tag expressed by the empty vector was prepared simultaneously.

Application of the recombinant protein in vivo

Shrimp were divided into four groups (10 individuals in each group). Three groups were injected with different amounts of rMjML1 (10, 3, and 1 μg/shrimp) and WSSV (5 × 105 copies per shrimp), and the other group was injected with the control tag protein (10 μg/shrimp) and WSSV (5 × 105 copies per shrimp). Thereafter, total RNA was extracted to determine the transcription level of WSSV VP28 using qRT-PCR. Protein samples were prepared to determine the translation level of VP28 using Western blotting with anti-VP28 Abs. Genomic DNA was also extracted to determine the virus copy number in shrimp tissues, following a previously described method (24). For the survival analysis, 30 shrimp were injected with WSSV (5 × 106 copies per shrimp) together with 5 μg of rMjML1 or control tag. The survival rates were recorded for both groups every 12 h for 72 h.

Western blotting

Shrimp gills were thoroughly homogenized in PBS, and the homogenate was centrifuged at 12,000 rpm × g for 20 min to collect the supernatant. The protein concentration was determined using the Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China). The supernatant was added to Protein Loading Dye (Sangon Biotech), boiled at 100°C for 5 min, and centrifuged at 8000 rpm × g for 3 min. The protein samples were separated by 10% SDS-PAGE with 100 μg protein loaded per lane. The proteins were transferred onto a nitrocellulose membrane using a conventional semidry transfer protocol with a constant voltage of 9 V for 50 min using a Jim-X Semi-Dry Blotter (Jim-X, Dalian, China). After blocking with 3% nonfat milk in TBS for 1 h, the membranes were incubated with specific primary Abs for 3 h at 25°C. After washing three times with TBST, the membrane was then incubated with HRP-labeled secondary Ab (1:10,000 diluted in nonfat milk) for 2 h. Unbound IgG was washed away using TBST. Bands were visualized using the High-sig ECL Western Blotting Substrate (Tanon Science & Technology, Shanghai, China). The chemiluminescent signal was detected using a 5200 Chemiluminescence Imaging System (Tanon Science & Technology). The anti-VP28, anti-Dorsal, and anti–β-actin Abs were made in our laboratory by immunizing New Zealand rabbits with the recombinant proteins (24–26). The anti-histone 3 Abs were purchased from Proteintech Group (Wuhan, China); all secondary Abs were purchased from Zhongshan (Beijing, China).

Separation of nuclear and cytoplasmic proteins

The separation of nuclear and cytoplasmic proteins was performed using a Nuclear Protein Extraction Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. Briefly, shrimp gills were washed with PBS three times and homogenized with 1 ml of cytoplasmic protein extraction reagent containing 1 mM PMSF. The homogenate was processed by five rounds of vortex shaking for 15 s and incubation in an ice bath for 3 min. The homogenate was centrifuged at 15,000 rpm × g for 25 min at 4°C, and the supernatant was collected as the pool of cytoplasmic proteins. The sediment was washed with PBS three times and then resuspended in nucleoprotein extraction reagent containing 1 mM PMSF. The suspension was processed by five rounds of vortex shaking for 15 s and incubation in an ice bath for 3 min and then centrifuged at 15,000 rpm × g for 25 min at 4°C. The supernatant protein was collected as the pool of nuclear proteins. The protein concentration was determined as described above.

Lipid-binding assay

The characterization of the binding of lipid cholesta-3,5-diene (CD) (Sigma-Aldrich, St. Louis, MO) to MjML1 was performed in three ways. An ELISA was performed as a preliminary binding assay. The 96-well plates were coated with 50 μl of CD or cholesterol solution (5 μg), incubated at 4°C until air dried for 3 d in a frost-free refrigerator, and blocked with 3 mg/ml of BSA solution. Purified rMjML1 was added to each well to a final concentration ranging from 5 to 100 nM. The plate was incubated at 25°C for 3 h and then washed with TBS three times. Mouse anti-His Abs (1:1000 diluted in 0.1 mg/ml of BSA solution) were added and incubated for 4 h at 25°C. Alkaline phosphatase–conjugated horse anti-mouse IgG (1:10,000 diluted in 0.1 mg/ml of BSA solution) was added after washing with TBS three times. After incubation for 3 h at 25°C, the wells were washed four times with TBS and added with p-nitro-phenyl phosphate (1 mg/ml in 10 mM diethanolamine with 0.5 mM MgCl2). The absorbance at 405 nm was then measured after incubation for 30 min at 25°C. Data are presented as the mean ± SD derived from three independent repeats.

The lipid-binding site of MjML1 was predicted by aligning MjML1 with human MD-2 and chicken MD-1. This short peptide was commercially synthesized by GenScript (Nanjing, China) at >95% purity. Using the synthesized peptide, isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) assays were performed to characterize the binding between MjML1 and CD in detail. The ITC assay was performed using MicroCal PEAQ-ITC (Malvern Panalytical, Malvern, U.K.). CD and the peptide were dissolved in 15% DMSO to a final concentration of 2 and 0.2 μM, respectively. According to the manufacturer’s instructions, CD was pumped in using a syringe, and the peptide was injected into the ITC cell. Injection of 2 μl of CD solution over a period of 150 s at a stirring speed of 750 rpm × g was performed. For the blank control test, CD solution was pumped into the sample cell of dissolvent. Before the analysis, the data were subtracted with that from the blank control test. A negative control test was performed as described above using an unrelated peptide comprising a partial peptide of Vibrio anguillarum flagellin A that was synthesized in the same way as the MjML1 peptide. This assay was repeated three times independently.

The SPR assay was performed using a BIACORE T200 (GE Healthcare Life Sciences, Chicago, IL). CD was coated onto the CM5 sensor chip using an Amine Coupling Kit (GE Healthcare Life Sciences). The synthesized peptide was dissolved to a series of final concentrations ranging from 40.625 to 1300 μM and injected to flow through the chip at a flow rate of 30 μl/min. Biacore Evaluation Software was used to calculate the association rates and dissociation rates using the one-to-one Langmuir binding model. The equilibrium dissociation constant was presented as the ratio dissociation rates/association rates. The SPR assay was performed independently three times.

Immunocytochemistry assay

Shrimp were injected with WSSV (5 × 105 copies), CD (1 μg), or rMjML1 (5 μg) with PBS, DMSO, or tag as the control. At specific time points after injection, the hemocytes were collected as described above. Fresh anticoagulant with 4% paraformaldehyde was used to wash and to incubate the hemocytes for 10 min. The hemocytes were then collected and resuspended in PBS and smeared onto poly-l-lysine–coated glass slides. One hour later, 0.2% Triton X-100 in PBS was added onto the slides and incubated for 10 min. After washing three times with PBS (for 6 min each time), the glass slides were blocked by 3% BSA in PBS at 37°C for 30 min. The hemocytes on the slides were then incubated overnight with anti-Dorsal Abs (1:500 in blocking buffer) at 4°C. After washing with PBS five times, the hemocytes were incubated with 3% BSA for 10 min. Then, goat anti-rabbit Alexa Fluor 488 (1:1000 diluted in 3% BSA) was added for 1 h in the dark. After washing with PBS five times, the hemocytes were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (AnaSpec, San Jose, CA) for 10 min at 25°C and then washed six times with PBS. Finally, the slides were observed under an Olympus DP71 fluorescent microscope (Olympus, Tokyo, Japan). The colocalization percentage of Dorsal- and DAPI-stained nuclei was analyzed using the Wright Cell Imaging Facility ImageJ software.

Chromatin immunoprecipitation assay

The chromatin immunoprecipitation (ChIP) assay was performed using a ChIP Assay Kit (Beyotime Biotechnology, Wuhan, China) according to the manufacturer’s instructions. Shrimp were injected with CD (1 μg) or DMSO. The hemocytes were collected 6 h after injection and used as the pool for ChIP. The primers used for the MjVago5 promoter are listed in Table I. The experiment was performed independently twice.

Statistical analysis

Most results in this study were analyzed using Student t test, and significance was accepted with p < 0.05. For the expression profiles, data were analyzed using one-way ANOVA, followed by Tukey multiple comparison test using GraphPad Prism software. For the survival assay, the results were analyzed using the log-rank (Mantel–Cox) test using GraphPad Prism software.

Results

Identification of the ML family from kuruma shrimp

Using the human MD-2 as a template, six proteins were identified as putative ML family members using local basic local alignment search tool searching from the data set of several previous transcriptomic analyses (J. Gao, J.X. Wang, and X.W. Wang, unpublished observations). Each protein contained a signal peptide and a typical ML domain, and they were named MjML1–MjML6 (GenBank accession numbers: MK993577, MK993578, MK993579, MK993580, MK993581, and MK993582). Multiple alignment of the shrimp ML family and mammalian ML proteins suggested that the cysteine residues responsible for the formation of disulfide bonds to stabilize the overall structure were conserved among all sequences (Supplemental Fig. 1). To reveal the possible relationship between the shrimp ML family and other typical vertebrate ML proteins, phylogenetic analysis was performed. As shown in Fig. 1, three subgroups of vertebrate ML proteins (MD-2 and MD-1, NPC2, and GM2A) were distinctly clustered, and the MjML family was separated into two clusters. MjML2, MjML3, MjML5, and MjML6 were clustered together with vertebrate NPC2 proteins, whereas MjML1 and MjML4 were clustered together with vertebrate MD-2 and MD-1 proteins. The relatively close relationship suggested that MjML 1 and MjML4 might play similar functions to vertebrate MD-2 and MD-1, which have been proven as important participants in innate immunity by recognizing LPS or other foreign lipid-like objects.

FIGURE 1.
FIGURE 1.

Neighbor-joining phylogenetic analysis of the ML family from kuruma shrimp (M. japonicus) with typical vertebrate MLs. The tree was built using MEGA 6.0 with bootstrapping of 1000. The accession numbers of the sequences used in the phylogenetic analysis are as follows: Homo sapiens NPC2, AAH02532; Mus musculus Npc2, AAH07190; Gallus gallus Npc2, NP_001026374; Xenopus laevis Npc2, AAH60392; Danio rerio Npc2, AAH45895; Ciona intestinalis Npc2, XP_002127695; M. japonicus ML5, MK993581; M. japonicus ML3, MK993579; M. japonicus ML6, MK993582; M. japonicus ML2, MK993578; G. gallus GM2A, XP_003642107; D. rerio Gm2a, XP_005173176; H. sapiens GM2A, AAD25741; M. musculus GM2A, CAJ18497; Rattus norvegicus GM2A, AAH72474; M. japonicus ML1, MK993577; M. japonicus ML4, MK993580; H. sapiens MD-1, AAC98152; M. musculus MD-1, BAA32399; G. gallus MD-1, NP_001004399; H. sapiens MD-2, BAA78717; Bos taurus MD-2, BAC67682; M. musculus MD-2, BAA93619; and Cricetulus griseus MD-2, AAK57984.

The shrimp ML family responded to WSSV infection and played antiviral roles

The tissue distributions of MjMLs transcripts were first analyzed (Table I). As shown in Fig. 2A, MjML1, MjML2, and MjML4 were widely expressed in all tested tissues, whereas MjML3 and MjML5 were expressed only in the hepatopancreas. To confirm the involvement of the ML family in WSSV infection, the expression profiles of MjMLs after virus infection were studied. The results showed a significant induction of both MjML1 and MjML3 in the hepatopancreas. MjML1 expression was induced more than 20-fold at the early stage of infection (6 h), whereas MjML3 expression gradually increased, reaching a peak at the mid and late stage of infection (24–72 h) (Fig. 2B). RNAi was then performed to knock down MjMLs expression to determine their specific roles during WSSV infection. As shown in Fig. 3A, MjML expression was successfully inhibited by dsRNA or siRNA injection. Knockdown of the expression of any MjML family member led to enhanced WSSV gene expression, with the highest enhancement occurring after knockdown of MjML1 or MjML3 (Fig. 3B). Taken together, these results suggested that MjML1 and MjML3 play important roles in restricting WSSV infection.

View this table:
Table I. Primers used for this study
FIGURE 2.
FIGURE 2.

Expression profiles of MjML genes. (A) Tissue distribution of MjML mRNAs. Total RNAs were extracted from healthy shrimp. Expression was studied using RT-PCR with β-actin as the internal reference. Each sample comprised at least five shrimp. The data are representative of two independent experiments. (B) Expression profiles of MjML mRNAs in the hepatopancreas after WSSV infection. qRT-PCR was performed to check MjML mRNA expression with β-actin as the reference. The expression level was normalized to the control test for each time point. The results are shown as the mean ± SD. Different characters indicate a significant difference, analyzed using one-way ANOVA, followed by Tukey multiple comparison test in GraphPad Prism. Repeats were performed in triplicate with at least five shrimp for each sample.

FIGURE 3.
FIGURE 3.

The function of MjML in WSSV infection, as analyzed using RNAi. (A) RNAi efficiency of MjML. dsRNA or siRNA (30 μg) was injected into shrimp hemocoel. RNAi efficiency was studied 48 h after dsRNA injection or 24 h after siRNA injection using qRT-PCR. (B) Expression of WSSV VP28 after MjML knockdown. WSSV infection (5 × 105 copies per shrimp) was performed 48 h after dsRNA or 24 h after siRNA injection. VP28 expression was checked another 24 h later using qRT-PCR. Three repeated experiments were performed independently. Each sample comprised at least five shrimp. Data show the mean ± SD. *p < 0.05, *p < 0.01, ***p < 0.001, as calculated using Student t test.

We decided to focus on MjML1. The enhanced WSSV infection after MjML1 knockdown was confirmed by observing higher levels of the of WSSV VP28 protein in the knockdown group compared with that in the control group (Fig. 4A). Recombinant MjML1 was then expressed, purified, and applied in vivo to further study its function in inhibiting virus infection (Fig. 4B). The results showed that rMjML1 application could suppress VP28 transcription (Fig. 4C) and VP28 translation (Fig. 4D) and reduced the viral titer (Fig. 4E) in shrimp tissue in a dose-dependent manner (Fig. 4C). Moreover, after shrimp were injected with rMjML1 or the control tag combined with WSSV inoculums, the survival rate of the rMjML1 group was always higher than that of the control group (Fig. 4F). Collectively, these data suggested that MjML1 plays an important role in the antiviral response.

FIGURE 4.
FIGURE 4.

Antiviral function of MjML1. (A) Expression level of WSSV VP28 protein after MjML1 knockdown. Protein samples were extracted 24 h postinfection in MjML1 preknockdown shrimp. VP28 protein expression was studied using Western blotting with anti-VP28 Abs. Data are representative of two independent repeats. (B) Recombinant expression and purification of MjML1 and control tag. rMjML1 and tag were expressed with recombinant and empty pET32a(+) vector in E. coli Rosseta (DE3) cells and purified using affinity chromatography. (C) Expression of WSSV genes after rMjML1 application in vivo. Shrimp were injected with different amounts of rMjML1 (10, 3, and 1 μg) and WSSV (5 × 105 copies). The control group was injected with tag (10 μg) and WSSV. WSSV VP28 expression was detected using qRT-PCR 24 h later. (D) Expression of the WSSV VP28 protein after rMjML1 application in vivo. Protein samples were extracted at 24 and 48 h postinfection and analyzed using Western blotting with anti-VP28 Abs. Data are representative of two independent experiments. (E) WSSV copy number quantification after rMjML1 application in vivo. Genomic DNA was extracted at 48 h after WSSV infection and rMjML1 application and was used to determine the WSSV copy number using qRT-PCR. At least five shrimp were used to prepare a sample. Three independent repeats were performed. Data are shown as the mean ± SD. **p < 0.01, ***p < 0.001, as analyzed using Student t test. (F) Survival rate after rMjML1 application. Shrimp were injected with rMjML1 or tag (5 μg) and WSSV (1 × 106 copies). Each group consisted of 30 shrimp. The survival rate was recorded every 12 h for 72 h. The results were analyzed using the Log-rank (Mantel–Cox) test in GraphPad Prism.

MjML1 recognized WSSV envelope lipid component CD

The mechanism by which MjML1 exerts its antiviral role was investigated. The close phylogenetic relationship between MjML1 and mammalian MD-2, which functions in the host response by recognizing nonself targets, prompted us to speculate whether MjML1 could bind any lipid components of WSSV, especially those of the WSSV surface envelope. A previous study found that although the WSSV envelope was generated from the host nucleoplasm, the cholesterol and hydrocarbons in WSSV were much more complicated than in the host hemocytes. Among the cholesterol and hydrocarbon-like molecules present in the WSSV envelope and absent in host nuclei, CD, the dehydroxyl derivative of cholesterol (Fig. 5A), accounted for the highest proportion other than cholesterol (27). Thus, we detected whether MjML1 could bind CD. As shown in Fig. 5B, rMjML1 preferentially bound to CD rather than cholesterol when coated onto the plates, and the binding increased as the dose of rMjML1 increased.

FIGURE 5.
FIGURE 5.

Binding of CD by MjML1. (A) Chemical structures of cholesterol and CD. (B) Binding of CD by rMjML1, analyzed using ELISA. CD or cholesterol (5 μg) was coated into 96-well plates and air dried. After blocking with 3 mg/ml of BSA, proteins (5 to 100 nM) were added into the wells for 3 h at 25°C. Bound proteins were detected using a conventional ELISA. Data show the mean ± SD. *p < 0.05, Student t test. (C) Identification of the lipid-binding site of MjML1. Alignment was performed using online ClustalW2 analysis. The β strands are labeled with arrows. The lipid-binding sites are in red. (D) Binding of CD by MjML1 lipid-binding peptide, analyzed using ITC. The ITC assay was performed with MicroCal PEAQ-ITC (Malvern Panalytical). CD (2 μM) was pumped in using a syringe, and peptide (0.2 μM) was injected into the ITC cell. Injection of 2 μl of CD solution over a period of 150 s at a stirring speed of 750 rpm × g was performed. Data shown are representative of three independent repeats. (E) Binding of CD by MjML1 lipid-binding peptide analyzed using SPR. SPR was performed with BIACORE T200 (GE Healthcare Life Sciences). CD was coated onto the CM5 sensor chip. Peptide solution (40.625 to 1300 μM) was injected to flow through the chip with a rate of 30 μl/min. This assay was performed independently in triplicate, and a representative result is shown.

ITC and SPR assays were then performed to study the binding properties of MjML1. The lipid-binding region of MjML1 was first identified by alignment of MjML1 with human MD-2 and chicken MD-1, whose lipid-binding properties had been revealed based on their crystal structures (Fig. 5C). The peptide encompassing the lipid-binding region of MjML1, located between β strands G and H, was commercially synthesized to meet the demand of high purity of protein in the ITC and SPR assays. Both the ITC (Fig. 5D) and SPR (Fig. 5E) assays confirmed the CD-binding ability of MjML1, with similar binding properties (dissociation constant) obtained (101 ± 23.1 μM in the ITC assay, 130.4 ± 29.86 μM in the SPR assay). These data suggested that MjML1 might exert its antiviral role by recognizing the lipid component of the WSSV envelope.

MjML1 regulated Vago5 expression after recognizing CD

We next attempted to identify the downstream effector molecule after MjML1 recognition of CD. We specifically focused on the Vago family, which was reported as the functional analogues of IFN in insect antiviral immunity (28). The Vago family is also expressed in L. vannamei and is important for shrimp antiviral immunity (29). Analysis of the transcriptomic data of kuruma shrimp and the sequences deposited in GenBank identified five Vago proteins in kuruma shrimp with the accession numbers BAW35380, BAW78900, BAW78901, BAW78902, and BAW78903. As shown in Fig. 6A, MjVago5 expression could be significantly induced by WSSV infection, suggesting its possible involvement in the antiviral response. Knockdown of MjVago5 expression using siRNA (Fig. 6B) led to a higher expression of WSSV VP28 than in the control group (Fig. 6C), confirming the significance of MjVago5 in the host–virus interaction.

FIGURE 6.
FIGURE 6.

Regulation of MjVago5 expression by CD-MjML1. (A) Induction of MjVago expression by WSSV. Shrimp were infected with WSSV (5 × 105 copies). Expression of MjVago was detected by qRT-PCR 24 h later. The fold induction was expressed as the ratio between the WSSV infection and the control group. The results show the mean ± SD from three independent repeats. (B) RNAi efficiency for MjVago5. siRNA (30 μg) was injected into shrimp. The expression of MjVago5 was detected 24 h later. (C) WSSV VP28 protein expression after MjVago5 knockdown. WSSV was injected into shrimp 24 h after MjVago5 siRNA injection. Protein samples were extracted at 24 and 48 h postinfection and analyzed using Western blotting with anti-VP28 Abs. Data are representative of two independent experiments. (D) Expression of MjVago5 after MjML1 knockdown or rMjML1 application. WSSV (5 × 105 copies) was injected 24 h after MjML1 siRNA injection (30 μg) or together with rMjML1 (5 μg). The expression of MjVago5 was detected 24 h later. Data are shown as the mean ± SD from three independent repeats. (E) The expression of MjML1 and MjVago5 after CD stimulation. Shrimp were injected with CD (1 μg) with DMSO as a control. The expression of MjML1 and MjVago5 was detected using qRT-PCR. (F) The effect of MjML1 on the induction of MjVago5 by CD stimulation. Shrimp were injected with CD (1 μg) 24 h after MjML1 siRNA injection (30 μg) or together with rMjML1 (5 μg). MjVago5 expression was detected 24 h later. The results are shown as the mean ± SD from three independent experiments. The results were analyzed using Student t test. *p < 0.05, **p < 0.001, ***p < 0.001.

The regulation of MjVago5 by MjML1 was then characterized. As shown in Fig. 6D, MjML1 knockdown obviously downregulated MjVago5 expression, regardless of the absence or presence of WSSV infection. Conversely, rMjML1 application increased the expression of MjVago5, further increasing the induction of MjVago5 by WSSV infection. Besides, CD significantly induced the expression of both MjML1 and MjVago5 (Fig. 6E). These data suggested that MjML1 transmitted the WSSV/CD signal to induce MjVago5 expression. To test this hypothesis, CD stimulation was performed in the shrimp in which MjML1 expression was presilenced. The results showed that CD-mediated induction of MjVago5 was suppressed by MjML1 silencing. Moreover, rMjML1 application enhanced CD-mediated induction of MjVago5 expression (Fig. 6F). This supported the view that MjML1 is a primary determinant for transferring the signal from CD to downstream MjVago5.

MjML1 regulated the subcellular location of the NF-κB transcription factor Dorsal

Mammalian MD-2 participates in LPS signaling by forming a receptor complex with TLR4 and activating NF-κB transcription factor–mediated inflammatory gene expression; therefore, we investigated whether MjML1 could function through regulating Dorsal, the principal NF-κB transcription factor of the arthropod Toll pathway. By separating and analyzing the cytoplasmic and nucleoproteins after WSSV infection or CD stimulation, obvious translocation of Dorsal from the cytoplasm to the nucleus was observed (Fig. 7A). This suggested that WSSV could induce the activation of the shrimp Toll pathway and that CD was indeed a molecular pattern for WSSV and was sufficient to imitate WSSV infection. As expected, rMjML1 application also led to Dorsal translocation, suggesting that MjML1 might regulate Dorsal function after recognizing the WSSV lipid component (Fig. 7A). To confirm the translocation of Dorsal after the above treatments, immunocytochemical analysis was performed to determine the distribution of Dorsal in shrimp hemocytes. As shown in Fig. 7B and 7C, WSSV infection, CD stimulation, and rMjML1 application could indeed induce the nuclear translocation of Dorsal. These data suggested that MjML1 regulates MjVago5 expression through Dorsal after immune recognition of WSSV/CD.

FIGURE 7.
FIGURE 7.

Induction of Dorsal translocation into nucleus by WSSV, CD, and rMjML1. (A) Distribution of the Dorsal protein after treatment, analyzed by Western blotting. Shrimp were injected with WSSV (5 × 105 copies), rMjML1 (5 μg), or CD (1 μg). Hemocytes were collected at specific times, and the cytoplasmic and nuclear proteins were separated. β-Actin and Histone3 were detected as references for the cytoplasmic and nuclear proteins, respectively. The data shown are the representative of two independent repeats. (B) Dorsal translocation analyzed using immunocytochemistry. Hemocytes were collected after specific treatments and fixed. The slides were analyzed using immunohistochemistry with anti-Dorsal Abs. Data are representative of two independent repeats. Scale bar, 10 μm. (C) Digitization of the result in (B). The colocalization percentage of Dorsal and nuclei was analyzed using WCIF ImageJ software. ***p < 0.001, as analyzed using Student t test.

CD stimulation enhanced the transcription of MjVago5 by Dorsal

The above results suggested an immune signaling transmission from WSSV/CD to MjML1, Dorsal, and finally, MjVago5. However, whether MjVago5 expression was directly or indirectly regulated by Dorsal remained uncertain. The promoter region of MjVago5 was cloned, and two NF-κB sites were identified in the promoter sequence (Fig. 8A). Next, a ChIP assay was performed to check whether Dorsal could bind the DNA fragment containing the NF-κB site. As shown in Fig. 8B, a positive signal was detected after immunoprecipitation using anti-Dorsal Abs with CD-stimulated hemocytes for the pool of ChIP. This suggested that Dorsal could directly transcriptionally regulate MjVago5 expression and that CD-stimulated translocation of Dorsal to the nucleus was essential for MjVago5 transcription. Thus, the above data supported the view that direct signal transduction from MjML1’s recognition of CD subsequently induced Dorsal translocation into the nucleus to regulate the expression of the antiviral effector MjVago5 (Fig. 9).

FIGURE 8.
FIGURE 8.

Regulation of MjVago5 transcription by Dorsal. (A) Analysis of the MjVago5 promoter. The 5′ untranslated region of MjVago5 was cloned and analyzed using the online Promoter Scan tool. (B) A ChIP assay was used to check the binding of Dorsal to the MjVago5 promoter. Shrimp were stimulated with CD (1 μg) or DMSO. The hemocytes were collected 6 h later as the pool for ChIP using a ChIP Assay Kit (Beyotime Biotechnology). Immunoprecipitates were used for RT-PCR with the primers designed against the MjVago5 promoter. The data are representative of two independent experiments.

FIGURE 9.
FIGURE 9.

Model of the MjML1-mediated antiviral mechanism. MjML1 recognizes WSSV by binding to its envelope lipid component, CD. MjML1 then transduces the viral signal to Toll4, leading to canonical TLR pathway activation and nuclear translocation of the NF-κB transcription factor, Dorsal. Dorsal transcriptionally regulates the expression and secretion of the antiviral molecule Vago, which activates the JAK/STAT pathway in neighboring cells.

Discussion

Vertebrate ML proteins can be clearly divided into three groups based on their sequence similarity, with the following proteins as the representative of each group: MD-2, NPC2, and GM2A. Accordingly, these three groups exert different functions in innate immune recognition and lipid metabolism (1). Compared with that in vertebrates, the ML family exhibited significant expansion in arthropods. Taking insects as examples, the A. aegypti genome encodes 24 ML proteins, the Anopheles gambiae genome encodes 13 ML proteins, and the Drosophila melanogaster genome encodes 8 ML family members (30). However, because of the high sequence variation of these proteins, it is difficult to separate the majority of them into distinct clusters like those in vertebrates. Moreover, most invertebrate ML proteins have not been functionally characterized, and thus, it might not be appropriate to simply name a certain ML protein only after its sequence homologue in mammals. Until now, only a few invertebrate ML proteins have been studied functionally. For example, among the eight ML proteins in D. melanogaster, Npc2a showed the highest sequence similarity with mammalian NPC2, and mutation of Npc2a led to abnormal cellular sterol distribution (31). The participation of Npc2a in sterol homeostasis and ecdysteroid biosynthesis suggested that it was indeed the ortholog of mammalian NPC2. However, together with other ML proteins in D. melanogaster, Npc2a also plays a role in innate immunity by binding bacterial polysaccharides and modulating the immune deficiency pathway, suggesting the complexity and significance of the ML family in invertebrate physiological processes (32). In the current study, we identified six ML proteins in kuruma shrimp M. japonicus. Through a phylogenetic analysis of the vertebrate ML family members, we preliminarily classified these six proteins as MD-2– and MD-1–like or NPC-2–like, based on the sequence information. This classification provided a clue for the subsequent functional analysis, which could in turn help to validate the classification.

Previous studies (21) and J. Gao, J.X. Wang, and X.W. Wang (unpublished data) supported the view that the ML family is important in WSSV infection. ML proteins could bind lipid ligands; therefore, two hypotheses of how shrimp ML proteins participate in WSSV infection could be proposed, referring to the data obtained for the mammalian ML family and for WSSV pathogenesis. First, the WSSV envelope originates from host cell nuclei; however, the lipid compositions and contents between them are somewhat different. In particular, there are more species of cholesterol and hydrocarbons in the WSSV envelope than in that of the host nuclei (27). Thus, the lipid material present in WSSV but absent in the host membrane might be recognized as a nonself target by ML proteins, which play an immune recognition function in this case. Second, WSSV enters host cells in a cholesterol-dependent manner (33, 34). The cholesterol-enriched lipid raft might provide a suitable microenvironment for virus attachment and endocytosis (35). Thus, ML proteins might regulate WSSV infection by influencing the transport of cholesterol. In the current study, we proved that MjML1 functioned in the antiviral response by recognizing the lipid component of WSSV. This MD-2–like immune recognition function was matched with the classification based on the sequence information. In addition, another WSSV-inducible ML protein, MjML3, also exerted an antiviral function. MjML3 is an NPC2-like molecule; therefore, further study should be performed to check whether MjML3 influences the WSSV infection by regulating lipid metabolism similarly to NPC2 in mammals.

Sequence analysis suggested that MjML1 adopted an overall canonical MD-2–like structure, with the conservation of multiple β strands. Several studies of the structures of MD-2 and MD-1 revealed that the lipid-binding site was located between β strands G and H (36, 37). We identified the lipid-binding site of MjML1 based on sequence alignment and successfully proved its interaction with lipids. Among MD-2–like proteins, there is a pronounced sequence variation of the lipid-binding sites, suggesting structural plasticity of the binding pocket to host lipid ligands. The discovery that MjML1 recognizes a WSSV lipid component and regulates antiviral molecule expression sheds new light on the mechanism of MD-2–like proteins in host–virus interaction. Among the limited reports of the relevance of MD-2–like proteins to virus infection, only one study found that human MD-2 could act as a receptor for the RSV F protein. By interacting with the RSV F protein in the hydrophobic pocket, MD-2 is essential for F protein–activated TLR4–NF-κB signaling (17). Our finding that MjML1 could directly bind WSSV lipids expanded the binding spectrum of MD-2–like proteins when acting as recognition proteins for viruses. To the best of our knowledge, this is the first report that the ML family directly recognizes the lipid components of viral pathogens.

Arthropod Vago proteins are regarded as functionally similar to vertebrate IFNs. Vago proteins contain a single von Willebrand factor type C domain and are secreted. The Vagos from D. melanogaster and Culex quinquefasciatus are induced by Drosophila C virus and West Nile virus (WNV), respectively, and control viral loads postinfection (28, 38). Moreover, C. quinquefasciatus Vago restricts viral infection by activating the JAK/STAT pathway to upregulate STAT-dependent virus-inducible molecules in neighboring cells. This IFN-like function was also proved in the white shrimp, L. vannamei (29). The results of the current study supported the importance of Vago in the antiviral response in kuruma shrimp. In addition, the finding that MjML1 induces shrimp Vago5 expression through the NF-κB transcription factor Dorsal increased our knowledge of the regulation of Vago expression. A previous study reported that induction of Culex Vago by WNV is dependent on Dicer-2, which is phylogenetically related to vertebrate RIG-I–like receptors and might sense viral nucleotides. Recognition of WNV by Dicer-2 leads to the activation of TRAF and the cleavage and nuclear translocation of Rel2, another NF-κB ortholog. Rel2 then binds the NF-κB site in the promoter of Vago, and this binding is essential for Vago induction (39). In the current study, kuruma shrimp Vago5 transcription was also mediated by NF-κB. However, the signal transduction upstream of NF-κB might be different in mosquito and shrimp. Both MD-2 and NF-κB factor are essential members of TLR pathway; therefore, it could be hypothesized that the recognition of viral lipids by MjML1 leads to the activation of the shrimp TLR pathway. A recent study showed that Toll4 in L. vannamei is critical for the defense against WSSV by inducing the nuclear translocation and phosphorylation of Dorsal, which then transcriptionally regulates the expression of several antiviral and antimicrobial peptides (40). However, the recognition events upstream of Toll4 remain unclear. Based on this information, a possible model could be proposed. MjML1 acts as a cofactor of Toll4 in kuruma shrimp. By recognizing the lipid component of the WSSV envelope, MjML1 senses viral infection and transduces the viral signal to Toll4. Through the canonical signaling of the TLR pathway, Dorsal is activated to transcriptionally regulate the expression of the antiviral molecule Vago5 (Fig. 9). The results of the current study revealed the significance of the ML family in antiviral response in kuruma shrimp. The identification of the CD–MjML1–Dorsal–Vago signaling pathway provided new insights into invertebrate antiviral immunity.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the National Science Foundation of China (Grants 31622058 and 31873043), the National Key Research and Development Program of China (2018YFD0900505), and the Young Scholars Program of Shandong University (Grant 2015WLJH26) (to X.-W.W.).

  • The sequences presented in this article have been submitted to GenBank under accession numbers MK993577, MK993578, MK993579, MK993580, MK993581, and MK993582.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CD
    cholesta-3,5-diene
    ChIP
    chromatin immunoprecipitation
    F
    fusion
    GM2A
    GM2 activator
    ITC
    isothermal titration calorimetry
    MD-2
    myeloid differentiation factor 2
    ML
    MD-2–related lipid-recognition
    NPC2
    Niemann–Pick disease type C2
    qRT-PCR
    quantitative real-time RT-PCR
    RNAi
    RNA interference
    RSV
    respiratory syncytial virus
    siRNA
    small interfering RNA
    SPR
    surface plasmon resonance
    WNV
    West Nile virus
    WSSV
    white spot syndrome virus.

  • Received March 4, 2019.
  • Accepted June 22, 2019.

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