Characterization of two novel ADP ribosylation factors from giant freshwater prawn Macrobrachium rosenbergii and their responses to WSSV challenge.

ADP-ribosylation factors (Arfs) are small GTP-binding proteins that have an essential function in intracellular trafficking and organelle structure. To date, little information is available on the Arfs in the economically important giant freshwater prawn Macrobrachium rosenbergii and their relationship to viral infection. Here we identified two Arf genes from M. rosenbergii (MrArf1 and MrArf2) for the first time. Phylogenetic analysis showed that MrArf1, together with MjArf1 from shrimp Marsupenaeus japonicus belonged to Class I Arfs. By contrast, MrArf2 didn't not match any of the Arfs classes of I/II/III, although it could be clustered with an Arf protein from M. japonicas called MjArfn, which may represent an analog of the Arf. MrArf1 was ubiquitously expressed in all the examined tissues, with the highest transcription level in the hepatopancreas, whereas MrArf2 was only highly expressed in the hepatopancreas and exhibited very low levels in the heart, stomach, gills and intestine. The expression level of MrArf1 in the gills was down-regulated post 24 h WSSV challenge, and reached the maximal level at 48 h. MrArf1 in the hepatopancreas went up from 24 to 48 h WSSV challenge. MrArf2 transcript in the gill also went down at 24 h and then was upregulated at 48 h WSSV challenge. MrArf2 increased significantly in the hepatopancreas 24 h after infection and then went down at 48 h WSSV challenge. RNAi results showed that knockdown of MrArf1 or MrArf2 could inhibit the expression of the envelope protein gene vp28 of the WSSV. So, it could be speculated that MrArf1 and MrArf2 might play important roles in the innate immune system against WSSV infection.

Introduction

Within years after its first discovery in Asia in the early 1990s, the white spot syndrome virus (WSSV) developed into an epizootic disease. WSSV is currently found in most crustacean farming areas in the world, where it causes large economic losses to the aquaculture industry. Aside from its economic effect, the disease also poses a threat to natural marine and freshwater ecology (Jiravanichpaisal et al, 2004, Nambi et al, 2012). As such, understanding the molecular mechanism of the innate immune defense against virus infection is imperative. Recent studies have demonstrated that the small GTPases found in shrimp participate in antiviral immunity by regulating phagocytosis (Liu et al, 2009, Wu et al, 2007). In Drosophila S2 cells (Schneider line 2), Ran protein, a small Ras-like GTPase, was involved in the regulation of phagocytosis against Drosophila C virus (DCV). Some members of the ADP ribosylation factors (Arfs) found in mammals (D'Souza-Schorey, Chavrier, 2006, Nie et al, 2003) and the shrimp Marsupenaeus japonicas (Ma et al, 2010, Wang et al, 2009, Zhang et al, 2010), which are classified with small GTP-binding proteins (~21 kDa), are involved in virus infection. The relationship between various Arfs and virus invasion is thus worth investigating.

Arfs are small, ubiquitously expressed GTP-binding proteins best known for their role in membrane trafficking (Beck et al, 2011, D'Souza-Schorey, Chavrier, 2006, Popoff et al, 2011, Sannerud et al, 2011) and in regulating actin cytoskeleton assembly (Myers and Casanova, 2008). The six mammalian Arfs are divided into three classes based on their amino-acid identity. Class I contains Arf1, Arf2, and Arf3; class II contains Arf4 and Arf5; and class III contains only Arf6. Class I and class II Arfs are mainly concentrated in the Golgi apparatus, although they also function in endosomal compartments. By contrast, Arf6 is primarily localized in the plasma membrane and is a subset of the endosomes. A large body of evidence indicates that Arfs promote the biogenesis of carrier vesicles by nucleating the assembly of coat protein complexes in the sites of vesicle formation (Cheung et al, 2014, Myers, Casanova, 2008). However, different Arfs seem to possess various intracellular localizations and functions, which depend on the specific membrane they bind according to their recruitment of diverse groups of proteins. Such specificities can be defined by determining which regulators of Arfs, GEFs (guanine nucleotide-exchange factors), and GAPs (GTPase-activating proteins) function with which Arf proteins (Donaldson, Honda, 2005, Sun et al, 2011). The binding of Arfs to membranes appears to be essential for viral replication (Belov et al., 2007a), and they cycle between GTP- and GDP-bound states. In the former cycle, the activated form Arf-GTP is bound to the membrane; in the latter cycle, Arf-GDP is released by the cellular GTPase activity into the cytoplasm.

During the evolutionary interplay between virus infection and host defense, the Arf-dependent secretion pathway may be involved in anti-virus action through secretion of interferons and presentation of viral antigens by MHC I (Ma et al, 2010, Molla et al, 1991). The pathway of secretion or endocytic can easily be made to cause viral immune evasion and virus propagation, and Arfs can be recruited to the specific target membrane to induce disintegration of intracellular membranes and formation of specific vesicles by viral proteins mediating their interactors (Belov et al., 2007b). Arf1 was reported to have important roles in the regulation of viral RNA replication and the production of infectious hepatitis C virus (HCV), a kind of plus-strand RNA virus (Matto et al., 2011). GBF1, a kind of Arf GEFs, was crucial for the RNA replication of coxsackievirus B3 (CVB3) (Lanke et al., 2009). In addition to the function in the viral RNA replication, class II Arf proteins (Arf4 and Arf5) played important roles in the secretion of dengue virus (DENV) (Kudelko et al., 2012). Mouse hepatitis coronavirus (MHV) replication was dependent on the GBF1-mediated Arf1 activation (Verheije et al., 2008). The replication of plant RNA virus also needed the participation of Arf1 (Hyodo et al., 2013). The previous research showed that Arfs were related to RNA virus replication and secretion; however, the relationship between Arfs and DNA virus infection was rarely reported. WSSV is a double stranded DNA virus and MjArf4 from M. japonicus was significantly up-regulated with the propagation of WSSV (Zhang et al., 2010). The relatively low expression level of MjArfn in WSSV-resistant shrimp may suggest its roles in WSSV invasion (Ma et al., 2010).

Compared with the extensive studies on shrimp Rab proteins (Attasart et al, 2009, Ongvarrasopone et al, 2011, Ye et al, 2012), Arfs were rarely studied. In this study, two Arf genes (MrArf1 and MrArf2) were identified for the first time from M. rosenbergii, an economically important crustacean in the aquaculture industry (Mohanakumaran Nair et al., 2006). The expression level of MrArfs were up-regulated with the WSSV propagation, which might preliminarily indicate the roles of MrArfs in WSSV infection.

Materials and methods

Animals, tissue collection and immune challenge by WSSV

M. rosenbergii (about 15 g each) were purchased from an aquaculture market in Zhenjiang, Jiangsu Province, China. The prawns were acclimatized in freshwater tanks under laboratory conditions at room temperature (25 °C). The prawns were divided into a control group and an experimental group designated as WSSV-challenged group. The WSSV inoculum was prepared following the previous paper (Wu et al., 2007). In brief, it was extracted from virus-infected M. japonicus by homogenizing muscle in TN buffer (20 mM Tris-HCl, 400 mM NaCl, pH 7.4). Then the tissue homogenates were centrifuged at 2000× g for 10 min. The supernatant was diluted 100 times with 0.9% NaCl and was subsequently filtered through 0.45 µm filter. About 100 µL of filtrate (105 WSSV copies/mL) was injected into the lateral area of the fourth abdominal segment of the prawns using a syringe with 29-gauge needle. The hepatopancreas and gills of the prawns challenged with WSSV at 0, 24, and 48 h (3 prawns at each time) were collected for RNA extraction. Tissues (i.e., heart, hepatopancreas, gills, stomach, and intestine) were also collected for RNA extraction.

Total RNA isolation, cDNA synthesis

The total RNA was extracted from the above-mentioned tissues using an EasyPure RNA Kit (TransGen, Beijing, China) according to the manufacturer's protocols respectively. The concentration of RNA were measured using Nanodrop (Thermo). For the qRT-PCR analysis, the first-strand cDNA was synthesized using a PrimeScript™ RT Reagent Kit (Perfect Real Time) (Takara, Dalian, China) with the primer oligo dT primer. For cloning the 3′ fragments of MrArfs, the first strand cDNA was synthesized using 3′-Full RACE Core Set Ver. 2.0 (Takara, Dalian, China) according to the manufacturer's protocol using 3′ RACE Adaptor primer. For cloning the 5′ fragments of MrArfs, 5′-RACE-Ready cDNA was produced using a Clontech SMARTer™ RACE cDNA amplification kit from TAKARA (Dalian, China). The detailed methods were according to the manufacturer's instructions with 5′-CDS Primer A (T25VN, N = A, C, G, or T; V = A, G, or C), SMARTer IIA oligo (AAGCAGTGGTATCAACGCAGAGTACXXXXX, X = undisclosed base in the proprietary SMARTer oligo sequence).

Cloning of the full-length cDNA of MrArf1 and MrArf2

Two EST fragments (MrArf1 and MrArf2) similar to Arf genes were found from the hepatopancreas transcriptome data. The EST sequences of the two genes were obtained from the hepatopancreas from M. rosenbergii using Illumina's Solexa Sequencing Technology performed by the Chinese National Human Genome Center at Shanghai. In order to obtain the full length of MrArf1 and MrArf2, the 3′ and 5′ RACE were performed. The 3′ ends of MrArf1 and MrArf2 were all cloned from the hepatopancreas using gene-specific primers (MrArf1-F: 5′- CAGTGTGGGATGTAGGTGGTC -3′ and MrArf2-F: 5′- TCAACGTCGAGACGGTGGAGC-3′) and 3′ RACE Outer Primer (5′-TACCGTCGTTCCACTAGTGATTT-3′). The 5′ ends of MrArf1 and MrArf2 were amplified using gene-specific primers (MrArf1-R: 5′-ATCACCAAAATCCCTCGGAAAACGC-3′ and MrArf2-R: 5′-AGGACCCTTGATTAGTGACAGGATGCCG-3′) and a Universal Primer A Mix (UPM: long, CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT; Short, CTAATACGACTCACTATAGGGC) according to the following procedures: 5 cycles at 94 °C for 30 s and 72 °C for 3 min; 5 cycles at 94 °C for 30 s, 70 °C for 30 s, and 72 °C for 3 min; and 25 cycles at 94 °C for 30 s, 68 °C for 30 s, and 72 °C for 3 min. The reaction was prepared in a Bioteke machine using a Clontech Advantage 2 PCR kit from Takara (Dalian, China). The full length of the MrArf1 and MrArf2 cDNAs were obtained by overlapping the 5′, middle, and 3′ fragments.

Sequence and phylogenetic analyses

Nucleotide sequence homology and comparisons of the deduced amino acid sequence were accomplished using the online program BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). cDNA translation and deduced protein prediction were obtained using ExPASy (http://www.au.expasy.org/). Signal sequence and domain prediction were performed using SMART (http://www.smart.embl-heidelberg.de/). MEGA 4.0 and GENDOC were used for the multiple alignments of MrArf1 and MrArf2 from M. rosenbergii. MEGA 4.0 was also used for constructing a phylogenetic tree and NJ (Neighbor-Joining) method was selected for the phylogenetic analysis (Kumar et al., 2008).

Real-time PCR

The tissue distribution of MrArf1 and MrArf2 at mRNA level in the heart, hepatopancreas, gills, stomach, and intestine was analyzed using Taqman probe qRT-PCR methods. The primers were (MrArf1-RT-F: 5′-CCAATCTCTTTAAGGGCCTTTTTG-3′, MrArf1-RT-R: 5′-TTTCACCCAATTTGAGCTTGTAAAG-3′, TaqMan fluorogenic probe: 5′-FAM-ACCAGCAGCATCTAACCCCACCA-Eclipse-3′; MrArf2-RT-F: 5′- CCCCTTGTGGAGGCATTATTTTC-3′, MrArf2-RT-R: 5′- CATTACGAGCAGAGGGCAGTAG-3′, TaqMan fluorogenic probe: 5′-FAM- CGTCCGCCGCCATCTTCATCGT -Eclipse-3′). The GAPDH from the prawns were also amplified as the reference gene using primers as follows (GAPDH–F: 5′'-AGGTCTTCAACGAGATGAAG-3′; GAPDH–R: 5′- GGAGTACTTCTCCAAGTTCA-3′; TaqMan fluorogenic probe: 5′-FAM- CAAGGCCGGAGCCGAGTACATC-Eclipse-3′). The time-course analysis of MrArf1 and MrArf2 in the hepatopancreas and gills of M. rosenbergii challenged with WSSV at 0, 24, and 48 h was performed using qRT-PCR methods with primers identical to those used during the tissue distribution analysis. A 2× Premix Ex Taq™ (Probe qPCR) (Takara, Dalian, China) was used in the qRT-PCR experiment following the manufacturer's instructions. The methods were undertaken as described by a previously published paper (Liu et al., 2009). All samples were repeated in triplicates in the qRT-PCR analysis. The data were calculated using 2−ΔΔCt methods (Livak and Schmittgen, 2001) and were subjected to statistical analysis. An unpaired sample t-test was conducted, and the difference was considered significant if P < 0.05.

RNA interference assay

DsRNA preparation: Gene-specific primers for MrArf1, MrArf2 and green fluorescent protein (GFP) were incorporated with the T7 promoter at their 5′-ends (RNAi-Arf1-F, 5′-GCG TAA TAC GAC TCA CTA TAG GAT ACT GGA GCC CAG TGT TC-3′; RNAi-Arf1-R, 5′-GCG TAA TAC GAC TCA CTA TAG GCT AAT ATA CAA GCT CCA CA-3′; RNAi-Arf2-F, 5′-GCG TAA TAC GAC TCA CTA TAG GGC ACT GTG ATC ACT TAA C-3′; RNAi-Arf2-R, 5′-GCG TAA TAC GAC TCA CTA TAG GAT CCT ATA AAT ACT TTC-3′; RNAi-GFP-F, 5′-GCG TAA TAC GAC TCA CTAT AGG TGG TCC CAA TTC TCG TGG AAC-3′; RNAi-GFP-R, 5′-GCG TAA TAC GAC TCA CTA TAG GCT TGA AGT TGA CCT TGA TGC C-3′). These primer sets were used to amplify the corresponding PCR products as templates for dsRNA synthesis. The dsRNA was synthesized using T7 polymerase (Fermentas, USA) following the previous method (Wang et al., 2009).

The Giant Freshwater Prawn was randomly divided into four groups. All Giant Freshwater Prawn in this assay were kept in tanks at about 22 °C. The first group was injected with 80 µg of dsMrArf1 RNA. The second group was injected with 80 µg of dsMrArf2 RNA. The third group was injected with 80 µg of dsGFP RNA. The untreated fourth group was used as control. Finally, all four groups were injected with WSSV (1 × 105 virions) at 48 h after dsRNA were injected. Total RNA were extracted from gills at 36 h after the WSSV injection. qRT-PCR analysis was used to detect the efficiency of the RNAi and WSSV levels. The primers of MrArf1 and MrArf2 were the same as part 2.5. Vp28 gene expression was detected using primers as follows (vp28-F: 5′-CACAGACAATATCGAGAC-3′; vp28-R: 5′- GCATCTTCTTCCTTCATC-3′; TaqMan fluorogenic probe: 5′-FAM- AACCTCCGCATTCCTGTGACT -Eclipse-3′). The experiments were repeated three times.

Results and discussion

Cloning of full-length cDNA of MrArf1 and MrArf2

Ran, Rab, and Rho proteins, members of small GTPases found in crustaceans, are involved in innate defense response (He et al, 2004, Liu et al, 2009, Pan et al, 2005, Wu et al, 2007). Arfs in mammalian cells participate in virus assembly, replication, and release (Belov et al., 2007a). Some of the Arfs of the shrimp M. japonicus are engaged in virus–host interaction events (Ma et al, 2010, Wang et al, 2009, Zhang et al, 2010). These findings suggest that investigating whether or not Arf GTPases in the giant freshwater prawn M. rosenbergii are involved in innate immunity against WSSV is noteworthy.

The sequencing of the transcriptome of the hepatopancreas and intestine of the freshwater prawns using the Solexa methods resulted in two different EST sequences similar to the Arf gene, which were designated as MrArf1 and MrArf2. The full-length cDNA of MrArf1 was 1819 bp, including a 132-bp 5′ untranslated region (UTR), a 549-bp open reading frame (ORF) encoding 182 amino acid proteins and a 1138-bp 3′ UTR. MrArf2 was 1364 bp in length with a 537-bp ORF encoding a protein with 178 amino acids. The 5′ and 3′ UTR of MrArf2 were 141 and 686 bp, respectively. The molecular weights of MrArf1 and MrArf2 were 20.7 and 19.9 kDa, respectively, and their pI values were 6.15 and 5.93, respectively. Each of them had an ARF domain (Supplement 1A and 1B). BLAST P analysis showed that MrArf1 was 100% identical with the ADP-ribosylation factor 1 from M. japonicus (MjArf1). However, the coding region cDNA sequence of MrArf1 was only 86% identical with that of MjArf1. The 3′ UTR of MrArf1 and MjArf1 were 64.3% identical to each other. In contrast to that of MjArf1, the 3′ UTR of MrArf1 had many extra insertions of nucleotides. Blast P analysis showed that MrArf2 shared 60% identity with MjArfn. The coding region of MrArf2 was 64% identical with that of MjArfn.

Multiple alignment and phylogenetic analysis of MrArf1 and MrArf2 with other Arfs

The multiple alignment of the MrArf1 with other Arf1s showed that the Arf1 proteins were highly conserved and had an N-terminal myristoylation site of G. The protein sequences of P loop (GLDAAGK), switch regions (GEIVT/ATIPTIGF, DVGGQDKIRPLWRHY), and interswitch region (NVETVEYKNISFTVW) from different Arf1s were nearly the same (Supplement 2A). MrArf2 also had an N-terminal myristoylation site of G. However, the protein sequences of P loop (GLDGVGKT), switch regions (GKVVQTIPTIGF, DIPSHSKMRPLWRHY), and interswitch region (NVETVEHGNISFTVW) of MrArf2 (Supplement 2B) were different from those of the MjArfn protein.

Phylogenetic analysis showed that the Arf proteins from different species could be divided into 4 different classes. Class I consists of Arf1, Arf2 and Arf3. MrArf1 from giant freshwater prawns and MjArf1 from shrimp M. japonicus belong to class I. Class II contains Arf4 and Arf5. MjArf4 and insect Arfs belong to class II. Arf6 proteins constitute class III. MrArf2 is not clustered into these 3 classes and MrArf2 and MjArfn belong to a unique class (Supplement 3).

The Arf proteins are ubiquitously expressed and well conserved in all eukaryotes with remarkable fidelity (D'Souza-Schorey, Chavrier, 2006, Li et al, 2004). The amino acid conservation among Arfs is striking, and the distinct clusters of class-specific residues may indicate unique interactions with regulators and effectors. Investigating these interactions will be challenging, but will reveal the full range of Arf functions in cells. Our results help lay the foundation for such future undertaking.

Tissue distribution of MrArf1 and MrArf2

qRT-PCR was used to examine the tissue distribution of MrArf1 and MrArf2. The results showed that MrArf1 was expressed in all analyzed tissues, including heart, hepatopancreas, gills, stomach, and intestine. The transcription level was significantly higher in the hepatopancreas than in the others (Supplement 4A). MrArf2 was only highly expressed in the hepatopancreas (Supplement 4B). MjArf1, MjArfn, and MjArf4 are all extensively expressed genes (Ma et al, 2010, Zhang et al, 2010). The characterization of the high conservation of protein sequences of shrimp Arfs and their constitutive and widespread expression in tissues may indicate that shrimp Arfs have important roles.

Transcriptional analysis of MrArf1 and MrArf2 after WSSV infection

Given our hypothesis that MrArf1 and MrArf2 are WSSV related, their gene transcriptional differential expressions during WSSV infection were evaluated. The results displayed a distinguished up-regulation of MrArf1 and MrArf2 post WSSV challenge, demonstrating that MrArf1 and MrArf2 can significantly be triggered by WSSV infection. Specific evidence was also presented to verify our hypothesis that MrArf1 and MrArf2 have a pivotal function in WSSV-related prawn innate immunity. However, MrArf1 and MrArf2 transcription patterns were distinct from those of Arf1, Arf4, and Arfn in the hepatopancreas of M. japonicus, which were up-regulated and quickly reached the highest level only 6 h after infection. In our study, MrArf1 reached the maximal level 48 h after infection (Fig. 1A and 1C ). The MrArf2 transcript in the gills was significantly down-regulated at first 24 h after infection (Fig. 1B) but up-regulated and reached the highest value at 48 h. In hepatopancreas, the MrArf2 was significantly up-regulated at 24 h after infection (Fig. 1D). These findings are consistent with the transcriptional tendency of Mjcent, the Arf6 GAP of M. japonicus (Wang et al., 2009). In the previous research, it was found that Arf1s and GBF1 were involved in the RNA virus replication and secretion (Kudelko et al, 2012, Matto et al, 2011, Verheije et al, 2008).

Fig. 1

Analysis of MrArf1 and MrArf2 expression in gills (A, B) and hepatopancreas (C, D) from the giant freshwater prawns M. rosenbergii challenged with WSSV using qRT-PCR methods.

MrArf1 and MrArf2 knockdown decreased the replication level of WSSV

In order to further indicate the roles of MrArf1 and MrArf2 in WSSV infection, RNAi experiment was performed. The results showed that MrArf1 and MrArf2 were knocked down in gills after MrArf1 and MrArf2 dsRNA were injected into the prawns (Fig. 2A and 2B ) and the envelope protein gene vp28 of the WSSV was obviously inhibited compared with the normal (untreated) control and the dsGFP injection group (Fig. 2C). Enterovirus 71 RNA replication was dependent on the activity of Class I Arfs and depletion of Arf1 and Arf3 inhibited viral replication in cells (Wang et al., 2014). In plants, RNA virus replication also needed the participation of Arf1 (Hyodo et al., 2013). Both the levels of hepatitis C virus (HCV) RNA and the production of infectious viral particles was downregulated when Arf1 expression was inhibited by small interfering RNA (siRNA) (Matto et al., 2011). Simultaneous depletion of class II Arf (Arf4 and Arf5) blocked dengue flavivirus secretion (Kudelko et al., 2012). The roles of Arfs in DNA virus infection was less reported. Centaurin-α1 is a shrimp GTPase activating protein (GAP) of Arf6 and it notably decreased in WSSV-resistant shrimps (Wang et al., 2009). Other member of Rab family, such as Rab27a, is required for human cytomegalovirus assembly (Fraile-Ramos et al., 2010). So, it could be speculated that MrArf1 and MrArf2 (members of Rab family) might play important roles in the innate immune system against WSSV infection.

Fig. 2

MrArf1 and MrArf2 knockdown decreases WSSV in giant freshwater prawn. The giant freshwater prawn was subjected to RNA interference with dsGFP as the control. (A, B) Effect of MrArf1 and MrArf2 RNAi on the target gene levels in the gills analyzed via qRT-PCR. (C) The giant freshwater prawn was infected with WSSV after MrArf1 and MrArf2 knockdown, and the amount of WSSV was detected via qRT-PCR using vp28 as a marker. Bars represent the mean of three individual measurements ± SEM. Differences between groups were analyzed using one-way ANOVA followed by a Tukey's multiple comparison test. Different letters indicate significant difference (P < 0.05).