White spot syndrome virus directly activates mTORC1 signaling to facilitate its replication via polymeric immunoglobulin receptor-mediated infection in shrimp

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture.


Author summary
White spot syndrome virus (WSSV) is the causative pathogen of white spot disease (WSD) and represents the most destructive viral disease of shrimp. The virus has evolved various strategies to escape from host defenses or exploit host biological pathways for its reproduction. Studies on viral immune-escape mechanisms can provide new strategies for disease prevention and control in shrimp aquaculture. Mechanistic target of rapamycin (mTOR) plays a central role in the regulation of cell growth and metabolism, which

Introduction
Mechanistic target of rapamycin (mTOR, also known as mammalian target of rapamycin) is a specific target protein of the drug, rapamycin, in eukaryotes [1]. Two mTOR homologs were originally identified in yeast [2], TOR1 and TOR2, whereas only one mTOR has been found to exist in other eukaryotes. mTOR belongs to the phosphatidylinositol-3 kinases (PI3K)-related kinase (PIKK) family and is an evolutionarily conserved serine/threonine protein kinase [3]. As an extremely important regulatory center in a variety of signaling pathways [4], mTOR controls several key biological processes, from protein synthesis to autophagy [5], mTOR has also manifold functions in regulation of innate immune responses in mammal [6]. mTOR nucleates two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which contain both unique and shared components. The two complexes differ regarding their upstream pathways, downstream targets, as well as in their structures and functions [7][8]. Moreover, mTORC1 regulates both the synthesis of a series of biological molecules (e.g., proteins, lipids, and nucleotides) [9], and strongly inhibits autophagy [10]. Thus, the role of mTORC1 in vivo is to simultaneously promote anabolic metabolism and inhibit catabolic metabolism [11], with its main function to promote cell growth. mTORC1 mainly regulates protein translation through its downstream key effectors, ribosomal protein S6 kinase (S6Ks) [12] and eukaryotic initiation factor 4E binding protein (4EBPs) [13][14] to control protein synthesis. Compared with mTORC1, the function of mTORC2 is less well studied. It has been shown that mTORC2 senses the stimulation of growth factors and plays a role in cell survival and actin reorganization [15][16].
Several studies have demonstrated that a variety of viruses are capable of activating, reducing, or suppressing the mTOR signaling pathway to support their own replication in hosts [17]. In addition, a number of viruses prefer to attack upstream targets of mTORC1, such as PI3K (Phosphoinositide 3-kinase) or AKT (protein kinase B), for viral replication (e.g., Epstein-Barr virus [EBV] [18] and human papillomaviruses [HPV] [19]), whereas other viruses prefer to act on downstream targets by promoting 4EBP1 and S6K1 phosphorylation. For example, the non-structural protein 5A (NS5A) of Hepatitis C virus binds to the mRNA cap-binding eukaryotic translation initiation 4F (eIF4F) complex and up-regulates host translation initiation machinery through phosphorylation 4EBP1 for translation of a select group of proteins beneficial to HCV infection [20]. While the majority of viruses hijack the mTOR signaling pathway, a subset of viruses can be inhibited by the mTOR pathway, such as the mosquito-transmitted bunyavirus, Rift Valley fever virus (RVFV). RVFV infection can induce translational arrest through the eukaryotic initiation factor 4E binding protein 1/2 (4EBP1/2) (target molecules of mTORC1)-dependent decay of 5'-terminal oligopyrimidine mRNAs and result in the restriction of viral infection [21]. A recent study found that mTOR plays an essential role via the positive regulation of RIG I-like receptor-mediated antiviral function in human dendritic cells [22].
White spot syndrome virus (WSSV) is the causative agent of white spot syndrome and represents the most destructive viral disease, responsible for substantial economic loss, a total of more than $7 billion in the shrimp industry [23]. Understanding the mechanisms of WSSV infection is of great importance for the prevention and control of the disease in shrimp. Previous studies have found that WSSV can utilize different receptors or attachment proteins for its infection in shrimp [24][25]. Among these, only two of the reported receptors are genuine transmembrane proteins: 1) beta-integrins [26]; and 2) polymeric immunoglobulin receptorlike protein (pIgR) [27]. As a WSSV receptor, pIgR in shrimp contains a signal peptide, an extracellular domain including an IG domain and two IG-like domains, a transmembrane region and an intracellular region. It mediates viral endocytosis via the pIgR-CaM-Catherin pathway [27]. mTOR signaling pathway is at the center of multiple signaling pathways. Indeed, previous studies have shown that following WSSV infection in shrimp, the PI3K-Akt-mTOR-HIF1α pathway is activated and promotes fatty acid synthesis and lipid metabolism, ultimately promoting viral replication in the shrimp host [28]. In addition, during WSSV infection, the mTOR signaling pathway was also activated to promote glycolysis via the PI3K-Akt-mTOR pathway and enhance viral replication in shrimp [29]. However, the mechanism by which mTOR signaling is directly activated by different viruses for their protein synthesis in invertebrates remains unclear. In the present study, we identified a mTOR in M. japonicus. By knocking down mTor or an injection of rapamycin, the inhibitor of mTORC1, we found that mTORC1 signaling provided a beneficial role to WSSV proliferation. We further elucidated that WSSV infection directly activated mTORC1 signaling via the VP24-pIgR-CaM-AKT signaling cascade.

WSSV exploits mTor to facilitate its replication in shrimp
The sequence, domain architecture, and phylogenetic analysis indicated that mTOR is a highly conserved protein in species ranging from shrimp to mammals (S1-S3 Figs). In addition, mTor is expressed in various tissues in shrimp, including hemocytes, heart, hepatopancrea, gills, stomach, and intestines in healthy shrimp (S4A Fig). We then investigated the mTor expression patterns at the mRNA level in shrimp challenged by WSSV. The results showed that mTor was significantly upregulated in the hemocytes (S4B To explore the function of mTor in shrimp infected with WSSV, mTor RNA interference (RNAi) was performed and WSSV replication was analyzed using the expression of Vp28 (the envelope protein gene of WSSV) and Ie1 (the immediate early gene of WSSV) as indicators. Following the knockdown of mTor in the hemocytes and intestines of shrimp infected with WSSV (Fig 1A), the levels of Ie1 and Vp28 mRNA expression were significantly decreased in these tissues (Fig 1B and 1C). The level of VP28 protein expression was also decreased in the shrimp compared with the shrimp injected with dsGfp ( Fig 1E and its statistical analysis 1e). Together, these results suggested that WSSV replication was significantly declined in the mTor-RNAi shrimp, which was also confirmed by the detection of WSSV copy numbers ( Fig  1D). We further analyzed the survival rate of the shrimp, and the results showed that the survival rate of mTor-RNAi group significantly increased following WSSV infection compared with the dsGfp-injected group (Fig 1F). These results indicate that mTor is utilized by WSSV to facilitate its proliferation in shrimp. mTORC1 signaling was activated by enhancing 4EBP1 phosphorylation in response to WSSV infection in shrimp mTOR functions via the multiprotein complexes, mTORC1 and mTORC2. To understand the effect of mTOR on the proliferation of WSSV via mTORC1 or mTORC2 signaling in shrimp, we first detected the phosphorylation of the 4EBP1, the target protein of mTORC1, in the hemocytes and intestines of shrimp following WSSV infection. The results revealed that 4EBP1 phosphorylation were significantly increased in the hemocytes and intestines of the shrimp infected by WSSV (Fig 2A and 2B), indicating that mTORC1 was activated in the WSSV-infected shrimp. Rapamycin, the inhibitor of mTORC1, was injected into the shrimp following WSSV infection, and Vp28 expression was analyzed as the indicator of the WSSV Genomic DNA was extracted from the tissues of mTor-knockdown shrimp infected with WSSV at 36 hpi. Significant differences were analyzed using a Student's t-test, P < 0.05 was considered to indicate a significant difference. (F) Survival rate of mTor-RNAi shrimp infected with WSSV. dsGfp injection was used as a control. The survival rate of each group was calculated, and the survival curves were presented as Kaplan-Meier plots. Differences between the two groups were statistically analyzed using a log-rank test in GraphPad Prism 8.0.  The toxicity of rapamycin in shrimp was detected using a shrimp viability analysis following a rapamycin injection (20 ng/g body weight). (E) Ie1 expression in the hemocytes proliferation. The shrimp were injected with different doses of rapamycin, and the results showed that Vp28 expression was significantly decreased in a dose-dependent manner in the shrimp (Fig 2C). Then the toxicity of rapamycin in the shrimp was further analyzed, the results showed that an injection with 20 ng/g rapamycin had no significant effect on shrimp mortality compared with the control group ( Fig 2D). We selected an injection of 20 ng/g rapamycin for all subsequent experiments. The results showed that the level of Ie1 and Vp28 expression were both decreased substantially in the rapamycin-injection group compared with the DMSOinjection group following WSSV infection (Fig 2E and 2F). The same results were obtained with a VP28 protein expression analysis (Fig 2G and 2g). The WSSV copy number was also markedly decreased in the gills and intestines of rapamycin-injection shrimp infected with WSSV ( Fig 2H). Furthermore, phosphorylation of 4EBP1 was significantly reduced in the shrimp injected with rapamycin following WSSV infection (Fig 2I and 2J). To further confirm that p-4EBP1 is regulated by mTORC1 in shrimp, we knocked down the specific target proteins in the mTORC1 and mTORC2 complex, Raptor and Rictor, and detected the phosphorylation level of 4EBP1. The results showed that following interference with Raptor by RNAi, the level of 4EBP1 phosphorylation was significantly decreased (S5C and S5c Fig). Finally, the survival rate of shrimp injected with rapamycin following WSSV infection was also analyzed. The survival rate of the shrimp increased significantly comparing with control ( Fig 2K). Collectively, the above results suggest that WSSV infection activates the mTORC1 signaling pathway to promote WSSV proliferation in shrimp.

WSSV exploits mTORC1 signaling for its proliferation via promoting the expression and phosphorylation of downstream target S6Ks
To verify the exploitation of mTORC1 signaling by WSSV for its infection, the expression of another downstream target in the signaling, ribosomal protein S6 kinases (S6K1 and S6K2 in S6A Fig), which are responsible for protein synthesis [30][31], were analyzed. We firstly detected the tissue distribution of S6k1 and S6k2, and found that S6k1 was distributed in all tested tissues, but S6k2 was hardly detected in hemocytes and stomach ( Fig 3A). Then the phosphorylation of S6K1 in shrimp challenged by WSSV was analyzed by western blot, the results showed that the phosphorylation of S6K1 increased significantly in the shrimp (Fig 3B), and phosphorylation of S6K1 was significantly inhibited in the shrimp injected with rapamycin both before and after WSSV infection ( Fig 3C). After knockdown of mTor, the level of S6k1 and S6k2 expression declined substantially ( Fig 3D). Next, S6ks-RNAi were performed to further explore the functions of S6k1 and S6k2 in WSSV-infected shrimp (Fig 3E and 3F), and results showed the levels of Ie1 and Vp28 mRNA were significantly decreased in the hemocytes and intestines of the shrimp compared with that of the dsGfp-injection group (Fig 3G and 3H). Moreover, VP28 expression was also analyzed at the protein level and the same results were and intestines of shrimp following an injection with rapamycin (20 ng/g body weight) analyzed by qPCR at 36 h post WSSV injection. (F) The level of Vp28 expression in the hemocytes and intestines of shrimp following rapamycin injection (20 ng/g body weight) analyzed by qPCR at 36 hpi. (G) The level of VP28 protein expression in the hemocytes and intestines of shrimp following an injection with rapamycin in shrimp; (g) Statistical analysis of the three independent experiments in panel G. (H) WSSV copy numbers in the gills and intestines of shrimp injected with rapamycin followed by WSSV infection. (I-J) Changes in the level of 4EBP1 phosphorylation in hemocyes (I) and intestines (J) of shrimp following treatment with rapamycin and WSSV infection. The upper panel represents the statistical analysis of the lower panel. The mRNA, protein and genomic DNA used for WSSV replication analysis were extracted from hemocytes and different tissues of the shrimp infected with WSSV at 36 hpi. (K) The survival rate of rapamycininjection shrimp following WSSV infection compared with the control group. The survival rate of each group was calculated, and the survival curves were presented as Kaplan-Meier plots. Differences between the two groups were statistically analyzed using a log-rank test in GraphPad Prism 8.0. Significant differences were analyzed using a Student's t-test, P < 0.05 was considered to indicate a significant difference; different lowercase letters indicate significant differences (P < 0.05) in the one-way ANOVA analysis. https://doi.org/10.1371/journal.ppat.1010808.g002

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication shrimp. An injection with dsGfp was used as a control. The mRNA and protein used for WSSV replication analysis were extracted from hemocytes and intestines of the shrimp infected with WSSV at 36 hpi. Significant differences were analyzed using a Student's t-test and significant differences were accepted at P < 0.05; different lowercase letters indicate significant differences (P < 0.05) in the one-way ANOVA analysis. https://doi.org/10.1371/journal.ppat.1010808.g003

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication obtained ( Fig 3I and statistical analysis 3i). These results suggest that protein synthesis through mTORC1 signaling is utilized by WSSV through activation of S6K1 and S6K2 in shrimp.

Inhibitor of AKT suppresses WSSV replication and 4EBP1 phosphorylation
To elucidate whether AKT, the upstream molecule of mTORC1, was involved in the activation of mTORC1 signaling in WSSV infection, an injection of MK2206, a specific AKT inhibitor, was administered, and activation of mTORC1 (using phosphorylation of 4EBP1 as the indicator) and WSSV replication (using Vp28 expression as an indicator) were analyzed. First, the toxicity of MK2206 in shrimp was analyzed, and the results showed that 1250 ng/g MK2206 injection had no significant effect on shrimp mortality ( Fig 4A). Next, we injected the shrimp with different doses of MK2206 following WSSV infection and detected the level of Vp28 expression. The results showed that Vp28 expression was significantly decreased in the shrimp at three different doses both in the hemocytes and intestines compared with the group injected with DMSO ( Fig 4B and 4C). The protein level of VP28 expression and AKT phosphorylation were both decreased in a dose-dependent manner in the MK2206-injection group compared with the DMSO-injection group following WSSV infection (Fig 4D and 4d). The WSSV copy number was also significantly decreased in a concentration-dependent manner in the intestines of shrimp injected with MK2206 ( Fig 4E). Next, we detected the levels of 4EBP1 and S6K1 phosphorylation following an injection with MK2206 (1250 ng/g). The results showed that the phosphorylation level of 4EBP1 and S6K1 were significantly decreased in hemocytes compared with the control group (Fig 4F and 4G). Finally, the survival rate of shrimp was also analyzed after injecting shrimp with MK2206 following WSSV infection, and the survival rate of the shrimp increased significantly (Fig 4H), All the results indicated that AKT was indispensable for the activation of mTORC1 signaling in WSSV-infected shrimp.

WSSV activates the mTORC1 signaling pathway via its receptor, pIgR mediated infection
Although several studies have reported that WSSV infection activates the mTOR signaling, it is not clear how WSSV activates this pathway. Thus, it is of interest to identify the viral factors involved in the activation of this pathway. Our previous research found that pIgR is a receptor of WSSV for viral infection via the pIgR-CaM-Catherin endocytosis pathway [27]. To further explore whether WSSV infection activates the mTOR signaling pathway through pIgR, pIgR-RNAi was performed and the expression of the mTORC1 target genes, S6ks, was analyzed. The results showed that after knocking down pIgR expression in the hemocytes and intestines ( Fig  5A), the level of S6k1 and S6k2 mRNA expression decreased significantly ( Fig 5B). We also analyzed phosphorylation of 4EBP1 (another mTORC1 target protein) and found that p-4EBP1 was significantly decreased in pIgR-RNAi shrimp following WSSV infection, and there were no obvious changes of p-4EBP1 in the shrimp without WSSV infection (Fig 5C and 5D). At the same time, the protein level of VP28 expression was significantly decreased in the hemocytes and intestines in pIgR-RNAi shrimp infected with WSSV ( Fig 5C and 5D). And we also detected the phosphorylation levels of S6K1 and AKT in the pIgR-RNAi shrimp and found the levels of S6K1 and AKT phosphorylation were significantly decreased in the pIgR-RNAi shrimp compared with the Gfp-RNAi group following WSSV infection, and there were no obvious changes in S6K1 ( Fig 5E) and AKT ( Fig 5F) phosphorylation in the pIgR-RNAi shrimp in the absence of WSSV infection. Meanwhile, we performed RNAi of β-Integrin (another WSSV receptor) [26] and detected the activation of mTORC1 by analyzing phosphorylation of 4EBP1 and WSSV replication in the shrimp. As is shown in S7 Fig  The survival rate of shrimp following an injection with MK2206 and WSSV compared with the control group. The survival rate of each group was calculated, WSSV replication was inhibited, p-4EBP1 level was not changed obviously in β-Integrin-RNAi shrimp. This result suggests β-Integrin mediated WSSV endocytosis did not use mTORC1 signaling for the viral proliferation. These results suggested that pIgR, as a receptor of WSSV, is involved in the activation of the mTORC1 signaling pathway in shrimp infected with WSSV.

The injection of recombinant VP24 activates mTORC1 signaling
The receptor, pIgR, interacts with the WSSV envelope protein, VP24, to mediate viral endocytosis [27]. To further confirm WSSV-mediated activation of mTORC1 signaling via the pIgR receptor, an injection of recombinant VP24 was performed, and the mTORC1 activation was analyzed by detecting expression and/or phosphorylation of mTOR, S6K1s, 4EBP1, and calmodulin (CaM). We first expressed VP24 and TRX-His-tag (for control) in Escherichia coli (S8A and S8B Fig) and removed endotoxin (LPS) with Triton X-114. Following an injection with the rVP24 or controls (rVP19 and TRX-His tag), the injected proteins in hemocytes were detected by immunocytochemical assays (S9A Fig), and the phosphorylation levels of 4EBP1 and S6K1 were also analyzed. The results showed that the level of 4EBP1 phosphorylation was significantly increased in the hemocytes and intestines of the rVP24 injection group compared with the control group, shrimp injected with TRX-His tag (Fig 6A and 6a), the level of S6K1 phosphorylation was also significantly increased in the hemocytes compared with the control group (S9B Fig). CaM can bind to pIgR and promote the entry of WSSV in shrimp [27]. Therefore, we detected the level of CaM protein expression. The results showed that CaM was significantly increased in the rVP24 injection group compared with the control group in the hemocytes and intestines (Fig 6B and 6b). To further corroborate the reliability of these results, we performed a similar experiment using rVP19 expressed in E. coli (S8C Fig), another WSSV envelope protein which does not bind to pIgR. We found that the level of 4EBP1 phosphorylation in shrimp did not change significantly following an injection with rVP19 ( Fig 6C and 6c), and there was also no significant change in phosphorylation of S6K1 (S9C Fig). In addition, the levels of mTor, S6k1, S6k2, and 4Ebp1 mRNA expression were detected after injection of rVP24 or rVP19 into shrimp for 24 h, the results showed that in the rVP24 injection group, the levels of mTor, S6k1, S6k2 and 4Ebp1 mRNA expression significantly increased compared with control group (Fig 6D), but no change in rVP19 injection group (Fig 6E). These findings indicated that similar to an WSSV infection, VP24 can activate mTORC1 signaling, however, rVP19 cannot activate mTORC1 signaling. Moreover, VP24 mediates the pIgR-CaM endocytic pathway to activate mTORC1 signaling in shrimp.

Calmodulin is involved in AKT phosphorylation and activation of mTORC1 signaling
It has been reported that CaM can bind to AKT and facilitate its translocation to menbrane, which is a critical step in its activation [32][33][34]. To explore the mechanism by which WSSV infection activates mTORC1 signaling, Cam-RNAi was performed, AKT and 4EBP1 phosphorylation was analyzed. Following a knockdown of Cam in the hemocytes and intestines (Fig 7A), The levels of AKT and S6K1 phosphorylation in the Cam-knockdown shrimp was inhibited in the shrimp infected with WSSV; however, there was no significant decline in the shrimp not infected with WSSV (Fig 7B and 7C). The phosphorylation of 4EBP1 was also and the survival curves were presented as Kaplan-Meier plots. Differences between the two groups were analyzed statistically using a log-rank test in GraphPad Prism 8.0. Significant differences were analyzed using a Student's t-test, P < 0.05 was considered to indicate a significant difference; different lowercase letters indicate significant differences (P < 0.05) in the one-way ANOVA analysis. https://doi.org/10.1371/journal.ppat.1010808.g004

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication significantly decreased in WSSV-infected shrimp. A reduction in the level of 4EBP1 phosphorylation was also observed in the WSSV-uninfected shrimp compared with the dsGfp-injection shrimp (Fig 7D). At the same time, the level Vp28 mRNA was significantly decreased in the hemocytes and intestines of the shrimp compared with that of the dsGfp-injection group ( Fig  7E). Next, we analyzed the interaction between CaM and AKT using a pulldown assay with the recombinant GST-tagged CaM and His-tagged pleckstrin homology (PH) domain of AKT (AKT-PHD) expressed in E. coli (S8D and S8E Fig). The results showed that CaM interacted with the pleckstrin homology (PH) domain of AKT in vitro, and there was no interaction between the GST tag with AKT ( Fig 7F). To further verify this interaction, we conducted isothermal titration calorimetry (ITC) to measure the Kd values for the binding of CaM to Akt-PHD. We obtained ITC data upon the titration of CaM into AKT-PHD under the same buffer conditions using a GST tag protein as a control. As shown in Fig

WSSV infection promotes viral protein translation and impedes host global protein translation in shrimp
Previous studies have shown that mTORC1 is the major regulator of protein translation in eucaryotes. Since viruses do not have their own translation machinery, the host's translation machinery is required for the translation of viral proteins [35][36]. To investigate the effect of WSSV infection on the global protein translation in shrimp, we labeled newly synthesized proteins with puromycin (S10A Fig), and then detected the global protein translation in shrimp by Western blot using an anti-puromycin monoclonal antibody. Different concentrations of puromycin were used to label the newly synthesized global proteins in shrimp. The content of the puromycin-labeled proteins increased with increasing concentrations of puromycin in shrimp (S10B Fig). Finally, 1 μg/g shrimp was used for protein labeling. The results showed that newly synthesized proteins in intestines of shrimp infected with WSSV were significantly lower than that of shrimp injected with PBS when VP28 was synthesized in a large number (S10C and S10c Fig). Together, these results suggest that an infection with WSSV can suppress the translation of host global proteins in shrimp and enhance the translation of the viral proteins probably via mTORC1 signaling, and this needs further study.
All of the above results suggest that WSSV infection directly activates mTORC1 signaling via the VP24-pIgR-CaM-AKT axis for viral proliferation in shrimp (Fig 8).

Discussion
In this study, we found that WSSV infection activated the mTORC1 signaling pathway to promote its replication via pIgR-mediated infection in shrimp. The WSSV VP24 protein was found to interact with pIgR and recruited CaM. The latter bound to the PH domain of AKT and induced AKT activation, which led to the activation of mTORC1 signaling and promoted WSSV proliferation via the downstream target proteins, 4EBP1 and S6K1, for viral protein upper panel represents the statistical analysis of three independent experiments of the lower panel. (E) The level of p-S6K1 in the hemocytes of pIgR-RNAi shrimp with or without WSSV challenge analyzed by Western blot at 36 hpi. The upper panel represents the statistical analysis of three independent western blot analyses. (F) The level of AKT phosphorylation in the hemocytes of pIgR-RNAi shrimp with or without WSSV challenge analyzed by Western blot at 36 hpi. The upper panel represents the statistical analysis of three independent experiments of the lower panel. In panel C-F, the subpanels of VP28/p-S6K1/pAKT and ACTB were from different gels with same amount of loading samples. Significant differences were analyzed using a Student's t-test, P < 0.05 was considered to indicate a significant difference; different lowercase letters indicate significant differences (P < 0.05) in the one-way ANOVA analysis. https://doi.org/10.1371/journal.ppat.1010808.g005

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication translation in shrimp. Our study further clarified that the viral factor, VP24, of WSSV was involved in the activation of this pathway through the pIgR receptor. In our opinion, this is the first study to reveal how WSSV directly activates mTORC1 signaling via its envelope protein, VP24, in animals.

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication mTORC1 plays a crucial role in promoting protein synthesis in eucaryotes [9,37]. Moreover, the translation of viral proteins in the host is an important process through which a virus can successfully infect a host. Several different viruses can hijack the mTORC1 signaling pathway and enhance downstream 4EBP1 phosphorylation and S6K1 activity to facilitate their own replication within the host [17]. For example, HPV infection induces increased 4EBP1 phosphorylation [21,38], and HCV infection directly promotes the phosphorylation of 4EBP1 [20]. In Litopenaeus vannamei, WSSV infection altered the host metabolome via the PI3K-Akt-mTOR pathway and achieved successful viral replication [29]. On the other hand, mTORC1 signaling can also restrict viral infection [21][22]39]. Previous studies have found that WSSV infection activates the PI3K-Akt-mTOR pathway to promote viral replication in hemocytes at the WSSV genome replication stage (12 hpi) by triggering aerobic glycolysis to provide energy and biosynthetic building blocks in shrimp [29]. WSSV infection can also use the PI3K-Akt-mTOR-HIF1α pathway to induce lipid biosynthesis at 24 h post-viral infection to support WSSV morphogenesis [28]. However, the mechanism by which WSSV activates the AKT-mTOR signaling pathway and which viral factors are involved in the activation of the mTORC1 pathway remain unknown. In our previous study, we found that WSSV utilizes GST and AKT (right). The lower panels show the ITC binding curves, indicating the amount of heat released per mole of CaM-AKT (left), buffer (middle), and GST-AKT (right). The calculated binding affinities (Kd) based on the ITC binding curves are shown in the figure. Significant differences were analyzed using a Student's t-test, and P < 0.05 was considered to indicate a significant difference. https://doi.org/10.1371/journal.ppat.1010808.g007

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication pIgR as a receptor for its infection through an interaction between its envelope protein, VP24. In addition, the pIgR intracellular domain recruits CaM to promote WSSV endocytosis into host cells [27]. Several studies have demonstrated that CaM can directly bind to the PH domain of AKT to induce the AKT translocation to the plasma membrane and ultimately promote the activation of AKT [32][33]40]. Activation of AKT in the plasma membrane is the key to initiating downstream AKT signaling pathways, including mTORC1 signaling [41]. To further explore how WSSV activates mTORC1 signaling, we knocked down the expression of pIgR, analyzed phosphorylation of 4EBP1 and S6K1, and found that 4EBP1 and S6K1 phosphorylation were inhibited in the pIgR-knockdown shrimp (Fig 5C and 5E). The same results were obtained following the knockdown of Cam (Fig 7C and 7D). Moreover, WSSV replication was also inhibited. We also found that CaM directly interacted with the PH domain of AKT (Fig 7F and 7G) and induced activation of AKT. The phosphorylated AKT activated mTORC1. Therefore, all of these results suggest that WSSV can directly activate mTORC1 signaling via the pIgR-CaM-AKT axis, and the WSSV envelope protein, VP24, is involved in activation of mTORC1 signaling. mTORC1 signaling promotes protein synthesis via the phosphorylation of two kinds of key effectors, S6Ks and 4EBP1. S6Ks are well-characterized targets of mTORC1. mTORC1 directly phosphorylates S6Ks, and subsequently S6Ks phosphorylates and activates several substrates that promote mRNA translation initiation [42]. In mammals, there are two homologs of S6K: S6K1 and S6K2 with high structural similarities and sharing redundant functions [30,43]. For example, the two S6K genes in mice seem to compensate each other, the expression of S6K2 in S6K1 knockout mice will increase, restoring the phosphorylation of rpS6 close to that of wildtype mice in the detected tissues [44]. Recent studies, however, challenge this notion, they may also exhibit distinct functions. For example, S6K2 regulates cancer cell survival via different routes [45]. In the present study, we have identified two homologous proteins of S6K, named S6K1 and S6K2 in M. japonicus (S6 Fig). We found that whether knockdown of S6k1 or S6k2, the replication of WSSV was both inhibited, and no obvious compensation of the two genes was detected. We also found no off-target effect of S6k1 RNAi and S6k2 RNAi (S6C and S6D  Fig). These might be that S6K1 and S6K2 regulate protein synthesis by different routes, which needs further study.
The 4EBP1 is the substrate of mTORC1, which is unrelated to S6Ks [14,46]. It inhibits translation by binding eIF4E to prevent assembly of the eIF4F complex. After phosphorylated by mTORC1, 4EBP1 triggers its dissociation from eIF4E [13,47], and promotes translation of 5'cap-dependent mRNAs, which involve in protein synthesis [48]. In our study, we found that WSSV infection increased phosphorylation of 4EBP1 (Fig 2A and 2B), knockdown of Raptor, the specific component of mTORC1 following WSSV infection, the phosphorylation of 4EBP1 decreased significantly (S5C Fig), and the proliferation of WSSV was decreased significantly (S5D Fig). These results suggest that as a downstream target of mTORC1, 4EBP1 involves in WSSV replication by promoting viral protein translation.
The results of the present study may indicate that mTORC1 is not the only signaling mechanism involved in WSSV replication. As shown in Fig 2E and 2F, the declined expression of Ie1 and Vp28, although showed significant difference, a moderate decrease was observed in the shrimp injected with rapamycin, the inhibitor of mTORC1 compared with control. This might suggest that inhibition of mTORC1 alone is not sufficient to reduce WSSV replication completely, and other signaling pathways (e.g., mTORC2) might be involved in WSSV replication.
Viruses can usurp the host translation machinery, targeting almost all steps in the process to that ensure viral protein synthesis is achieved for viral replication [49]. Some viral proteinases can interact with host proteins (i.e., host translation machinery proteins) to inhibit host protein translation [50]. For example, caliciviruses can encode proteases that cleave eIF4G to suppress cap-dependent translation [51]. Other viruses depend on cap-dependent translation and stimulate eIF4F activity by promoting mTOR and eIF4E phosphorylation, as well as the assembly of translation machinery [49]. One recent study found that during Newcastle Disease virus (NDV) infection, viral mRNAs are efficiently translated, whereas host cellular protein synthesis is almost completely inhibited. This effect was attributed to the ability of NDV to activate the PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to promote viral mRNA translation via interactions between the viral NP protein and host eIF4E [52]. Similar results were obtained in the present study; there was an observed inhibition of host protein translation and a significant increase in the translation of viral proteins in shrimp following WSSV infection (S10 Fig). The possible mechanisms by which WSSV mediates these effects require further study.
In summary, we identified the VP24-pIgR-CaM-AKT-mTORC1 signaling cascade as a novel pathway by which WSSV facilitates its proliferation in shrimp (Fig 8). WSSV entered host cells via the binding of pIgR through VP24 and inducing the interaction between the pIgR intracellular domain with CaM [27]. Upon interacting with the PH domain of AKT, CaM promoted the phosphorylation of AKT, thereby activating the downstream mTORC1 signaling pathway to promote viral DNA and protein synthesis through 4EBP1, S6K1 and S6K2, the target proteins of mTORC1 in shrimp.

Ethics statement
The rabbit experiments for antibody preparation in the study were carried out in accordance with protocols approved by the Animal Care & Welfare Committee at Shandong University School of Life Sciences (SYDWLL-2021-53).

Animals
Healthy M. japonicus (9 g-12 g each) were purchased from a market in Qingdao, Shandong, China. The animals were cultured in a laboratory shrimp culture system with natural sea water for at least 24 h to acclimate the culture system. The animals were randomly selected for the following experiments. Each of the sample was extracted using tissues from 3-5 shrimp and all the experiment were performed three times independently.

WSSV challenge and tissue collection
The WSSV inoculum was prepared based on previously described methods and quantitative real-time PCR (qPCR) was used for viral quantification [53]. Each shrimp was injected with 50 μL WSSV virions (1 × 10 6 ) for infection. The same volume of sterile phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , pH 7.4) was injected into the control groups. Hemolymph was extracted from 3 or 4 shrimp using a sterile syringe with anticoagulant buffer (450 mM NaCl, 10 mM KCl, 10 mM EDTA, 100 mM HEPES, pH 7.45) and centrifuged at 800 × g for 6 min at 4˚C. Hemocytes were collected for further experiments. Other tissues were dissected with scissors and forceps on ice from at least three shrimp for RNA or protein extraction.

RNA extraction, cDNA synthesis, DNA and protein extraction
Total RNA was isolated from the hemocytes and different organs (heart, hepatopancreas, gills, stomachs, and intestines) of shrimp using TRIzol (ET101, Transgen, Beijing, China). First strand cDNA synthesis was performed using a cDNA Synthesis Kit (5×All-in-One RT Master-Mix; Applied Biological Materials-abm, Vancouver, Canada) in accordance with the manufacturer's instructions. Genomic DNA was extracted using a Genomic DNA Purufication Kit (Toyobo, Osaka, Japan). Protein samples from different organs and hemocytes were obtained by separately homogenizing in radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% EDTA, 50 mM Tris-HCl, pH 7.8). The homogenates were centrifuged at 12000 × g for 10 min at 4˚C and the supernatant was collected for further analysis.

Tissue distribution and mTor expression profiles
The total cDNA sequences for mTor from the shrimp were obtained from the transcriptome sequencing of M. japonicus [54]. The tissue distribution of mTor mRNA was determined by semi-quantitative reverse transcription-PCR (RT-PCR) using mTor-RT-F and mTor-RT-R primers (S1 Table). β-actin was used as the internal control, with β-actin-RT-F and β-actin-RT-R primers. The PCR procedure consisted of an initial incubation at 94˚C for 3 min; followed by 32 cycles of 94˚C for 30 s, 52˚C for 30 s, and 72˚C for 30 s; followed by 72˚C for 10 min. The PCR products were analyzed using agarose gel electrophoresis (1.5% agarose).
Quantitative real-time PCR (qPCR) was performed to determine the expression profiles of mTor in the shrimp following a challenge with WSSV using the above primers. qPCR was performed as follows: 95˚C for 10 min; 40 cycles at 95˚C for 10 s, and 60˚C for 50 s; followed by a melting period from 65˚C to 95˚C. The obtained data were analyzed using the cycle threshold (2 −ΔΔCT ) method [55]. β-actin and the elongation factor-1α (Ef-1α) were used as the internal controls with primers β-actin-RT-F, β-actin-RT-R and Ef-1α-F, and Ef-1α-R, respectively. The results were expressed as the mean ± SD from three independent replicates.

Bioinformatic analysis
The total cDNA sequences for mTor, Akt, S6k1, S6k2, and 4Ebp1 were all obtained from the transcriptome sequencing of M. japonicus and the sequences were confirmed by RT-PCR. The amino acid sequences, theoretical molecular weights, and isoelectric points of the above molecules were analyzed using an online server (http://web.expasy.org/translate/). A domain prediction tool (SMART: http://smart.emblheidelberg.de/) was used to analyze the protein domain architecture. Phylogenetic trees of the molecules from different species were constructed using MEGA7 (Download from https://www.megasoftware.net/).

RNA interference (RNAi)
RNAi was performed to analyze the function of mTOR. Gene-specific primers dsmTor-F and dsmTor-R were linked to the T7 promoter (S1 Table) and used to amplify a partial sequence of mTor cDNA. The PCR products were used as the templates for double-stranded RNA (dsRNA) synthesis using T7 RNA polymerase (Fermentas, Burlington, Canada), following the manufacturer's instructions. The dsGfp (green fluorescent protein) coding region, serving as a control, was amplified using the dsGfp-F and dsGfp-R primers (S1 Table) and synthesized using the above method. For the RNAi assay, shrimp were randomly divided two groups (20 shrimp/group), and dsmTor (5 μg/g) was injected into the muscles of shrimp using a 50 μL syringe. The same dose of dsRNA was injected again at 12 h following the first injection. The same dose of dsGfp was used as a control. RNAi efficiency was assessed after 24 h using qPCR. Similar methods were used to knockdown Raptor, Rictor, S6k1, S6k2, pIgR, Cam and β-Integrin.

Rapamycin injection
The mTOR inhibitor, rapamycin (Aladdin, Shanghai, China) was dissolved in DMSO to create a highly concentrated storage solution (10 mg/mL). When using, the storage solution was first diluted to 40 μg/mL with DMSO, and finally, 4 μg/mL of the required injection solution was diluted with PBS. The shrimp were divided into two groups (15 shrimp/group). The rapamycin solution (50 μL) was injected into each shrimp at the penultimate segment. The final concentration of the rapamycin solution injected into the shrimp was approximately 20 ng/g shrimp. The control group was injected with same volume of PBS containing 10% DMSO.

Survival rate assay
The survival rate was analyzed following mTor-RNAi in shrimp challenged with WSSV. The shrimp were divided into two groups (40 shrimp/group): 1) dsGfp group; and 2) dsmTor group. After administering RNAi for 24 h, an inoculum of WSSV (1 × 10 5 copies) was separately injected into two groups of shrimp. The number of dead shrimp in the two groups were observed every 12 h after infection, the survival rate of each group was calculated, and the survival curves were presented as Kaplan-Meier plots. Differences between the two groups were analyzed with a log-rank test using GraphPad Prism 8.0 software. Significant differences were considered at a threshold of P < 0.05.
The survival rate of the rapamycin-injected shrimp was also analyzed. The shrimp were randomly divided into two groups (35 individuals/group): 1) the rapamycin-injected group; and 2) the DMSO-injected group. At 2 h following rapamycin injection, WSSV (1 × 10 5 ) was separately injected into two groups of shrimp. The number of dead shrimp in the two groups was observed every 12 h after infection, and the dead shrimp were immediately removed. The survival rate was obtained using the above method.

Phosphorylation analysis of 4EBP1 and S6K1 in shrimp following WSSV infection
To explore whether mTORC1 is activated by WSSV, phosphorylation of 4EBP1 and S6K1 were analyzed by Western blot as an indicator. The shrimp were randomly divided into two groups (20 shrimp each group): one group was injected with WSSV (1 × 10 6 ), and a control group was injected with same volume of PBS. Hemocytes and intestines were dissected from the shrimp in each of the two groups at different time points for protein extraction. Western blotting was used to analyze the phosphorylation of 4EBP1 and S6K1 in shrimp using a p-4EBP1 antibody and p-S6K1 antibody.

Detection of global protein translation in shrimp
As a peptidyl transfer inhibitor, puromycin has been widely used in cell biology to tag newly synthesized proteins [56]. We used puromycin (MACKLIN, Shanghai, China) to analyze the nascent proteins in shrimp challenged by WSSV and PBS. Shrimp were divided into two groups (10 individuals /group), and WSSV (1 × 10 6 ) or PBS were injected into shrimp. Proteins were extracted from the shrimp at 12, 24, and 48 h post-injection with WSSV and PBS. One hour before protein extraction, the shrimp in the experimental and control groups were injected with the same dose of puromycin (1 μg/g). A Western blot analysis was performed and puromycin integration into the polypeptide was detected using a puromycin monoclonal antibody. The degree of puromycin integration into the polypeptide reflected the level of global protein translation in the shrimp following the injection with WSSV and PBS.

Expression and purification of recombinant proteins
The recombinant proteins were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 37˚C for 4 h for expression. The GST-tag proteins were purified using affinity chromatography with GST-resin (GenScript, Nanjing, China) in accordance with the manufacturer's instructions. Recombinant His-tagged proteins were purified by high-affinity Ni-IDA Resin (Gen-Script). In addition, for the proteins used in vivo (e.g., His-TRX-tag, rVP24, and rVP19), an additional wash in cold 0.1% Triton X-114 was performed to remove any endotoxin contamination [57].

Pull-down assay
A pull-down assay was performed to explore the interaction between calmodulin (CaM) and AKT protein kinase B. CaM was recombinantly expressed in E. coli using the recombinant vector, pGEX-4T-1/CaM, and the PH domain of AKT was recombinantly expressed in E. coli with recombinant vector pET-32a/AKT-PHD. Purified GST-tagged CaM (200 μg) was incubated with the His-tagged AKT-PHD (1:1) overnight at 4˚C. Following an incubation with GST-bound resin (100 μL) for 50 min at 4˚C, the resin was washed five times with PBS. Elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0) was added to elute the bound proteins. SDS-PAGE was conducted for protein analysis. A GST-tagged protein was used as a control.

Isothermal Titration Calorimetry (ITC)
The thermodynamic parameters for CaM binding to AKT-PHD were determined using an Auto-iTC200 microcalorimeter (Malvern Panalytical, Malvern, UK). ITC experiments were performed at 25˚C, and the protein samples were suspended in 0.1M Tris-HCl (pH 8.0) buffer. CaM (70 μM) was titrated into a sample cell containing 10 μM AKT-PHD. The heat of the

PLOS PATHOGENS
WSSV directly activates mTORC1 signaling to facilitate its replication reaction was measured at 25˚C for the 19 injections by titrating CaM into the buffer. Data analysis was performed using the Malvern MicroCal PEAQ-ITC Analysis. Baseline corrections were performed by subtracting heat of dilution for the binding of CaM to AKT-PHD. The binding curves were analyzed, and dissociation constants (Kd) were determined by nonlinear least square fitting of the baseline-corrected data.

MK-2206 2HCl injection
The AKT inhibitor, MK-2206 2HCl (AbMole, America), was dissolved in DMSO to make a highly concentrated storage solution (5 mg/mL). The storage solution was first diluted to 2.5 mg/mL with DMSO and finally, the 0.25 mg/mL required injection solution was diluted with PBS. The shrimp were divided into two groups (10 shrimp/group) and the MK-2206 solution (50 μL) was injected into the body at the penultimate segment of the shrimp, after 2 h of inhibitor injection, WSSV (1 × 10 6 ) was injected into two groups of shrimp. The final concentration of the MK-2206 solution that was injected into the shrimp was approximately 1250 ng/g shrimp. The control group was injected with PBS containing 10% DMSO.
The survival rate of the MK-2206-injected shrimp was also analyzed. The shrimp were divided into two groups (35 shrimp/group): 1) the MK-2206-injected group; and 2) the DMSO-injected group. At 2 h following the injection with the inhibitor, WSSV (1 × 10 5 ) was separately injected into two groups of shrimp. The number of dead shrimp in the two groups was observed every 12 h post-infection, and the dead shrimp were immediately cleaned up. The survival rate was obtained using the methods as described above.

Statistical analysis
Data were presented as the mean ± standard deviation (SD) of at least three replicates for statistical analysis. Significant differences were analyzed using two-tailed Student's t-test for paired comparisons or a one-way ANOVA for multiple comparisons. P value < 0.05 was considered statistically significant. Different lowercase letters indicate significant differences (P < 0.05) in the one-way ANOVA analysis. The survival rate was calculated, and the survival curves are presented as Kaplan-Meier plots and the statistically using a log-rank test. All statistical analyses were produced using GraphPad 8.0 data view software. Densitometry analyses of Western blot bands were based on three independent replicates using ImageJ software (National Institutes of Health, http//imagej.nih.gov/ij/download.html). and Ie1 (C) at the transcriptional level detacted by qPCR. D. The WSSV copy number decreased significantly after β-Integrin knockdown. E. Phosphorylation of 4EBP1 was detected after knocking down of β-Integrin. e. Statistical analysis based on three independent experiments of (E). Significant differences were analyzed using a Student's t-test, and P < 0.05 was considered to indicate a significant difference. experiments. Significant differences were analyzed using a Student's t-test, and P < 0.05 was considered to indicate a significant difference. (TIF) S1 Table. Primer sequences used in this article. Annealing temperature (˚C). Amplicon size (bp). The amplification efficiency of the primers (%) (DOCX)