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, 12 (12), e1006127
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Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp

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Scavenger Receptor C Mediates Phagocytosis of White Spot Syndrome Virus and Restricts Virus Proliferation in Shrimp

Ming-Chong Yang et al. PLoS Pathog.

Abstract

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. MjSRC was upregulated in shrimp challenged by WSSV.
(A) Tissue distribution of MjSRC in shrimp. The mRNA expression level was analyzed using qRT-PCR (upper panel). The protein expression level was detected by western blotting (lower panel). β-Actin was used as the internal reference. (B) MjSRC-EX recombinant expression in E. coli, analyzed by SDS-PAGE, and MjSRC in hemocytes of shrimp, detected by western blotting using an anti-MjSRC sera as the primary antibody. (C) Expression patterns of MjSRC mRNA (upper panel) and protein (lower panel) in hemocytes of shrimp after WSSV challenge, detected by qRT-PCR and western blotting with the β-actin gene as the reference. Results were expressed as mean ± SD and analyzed statistically by Student’s t-test. *, p < 0.05, **, p < 0.01 and ***, p < 0.001. (D) The protein expression pattern of MjSRC was digitalized using Quantity One software by scanning the western blotting bands from three independent repeats. Relative expression levels of MjSRC/β-actin were expressed as the mean ± SD, and the value of the normal shrimp at 0 h was set as 1. Significant differences were analyzed by Student’s t-test. (E) VP28 expression levels analyzed by western blotting to detect WSSV replication in the gill. β-Actin was used as the sample loading control. (F) Expression patterns of MjSRC mRNA in hemocytes of shrimp after UV-WSSV challenge, detected by qRT-PCR with the β-actin gene as the reference. Results were expressed as mean ± SD and analyzed statistically using Student’s t-test.
Fig 2
Fig 2. MjSRC restricts WSSV replication in shrimp.
(A) Agarose gel electrophoresis of in vitro amplified or synthesized cDNA, mRNA and dsRNA of MjSRC, used in subsequent overexpression and RNAi assays. (B) Efficiency of MjSRC RNAi in hemocytes, as detected by qRT-PCR (upper panel) and western blotting (lower panel). (C) Efficiency of MjSRC overexpression in hemocytes, as detected by western blotting with anti-MjSRC sera (lower panel). The protein expression level of MjSRC was digitalized using Quantity One software by scanning the western blotting bands from three independent repeats (upper panel). (D) WSSV replication in shrimp after overexpression of MjSRC. The shrimp was injected with WSSV after MjSRC mRNA injection. The VP28 expression in gills was determined at 48 h after WSSV injection using qRT-PCR (upper panel) and western blotting (lower panel). Trx-His tag mRNA overexpression was used as the control. (E) WSSV replication in MjSRCi-shrimp and MjSRC-rescue shrimp. Shrimp were divided into five groups, and WSSV replication was detected using qRT-PCR (upper panel) and western blotting (lower panel). Differences between each group were analyzed using one-way ANOVA. Different letters indicate statistical significance (p < 0.05). β-Actin was used as the internal reference. (F) The quantification of virion copies in gills from each individual shrimp in the five groups detected by qRT-PCR using the standard curve. Eight shrimp were used in each group. Differences between each group were analyzed using one-way ANOVA. Different letters indicate statistical significance (p < 0.05) and the same letter indicate no statistical difference (p > 0.05). (G) The survival rate of MjSRC-RNAi shrimp infected with WSSV. Shrimp were divided into two groups (40 shrimp in each group). After 24 h of dsRNA injection, WSSV inoculums were injected. Shrimp survival was monitored every day after WSSV injection. dsGFP injection was used as the 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 analyzed with log-rank test using the software of GraphPad Prism 5.0. p = 0.0021.
Fig 3
Fig 3. MjSRC enhances the phagocytosis of WSSV in hemocytes of shrimp.
(A-C) Phagocytic analysis after RNAi of MjSRC. (A) Hemocyte phagocytosis observed under the fluorescence microscope. WSSV virions were labeled with FITC (green) and then injected into shrimp. The hemocytes from three to five shrimp were collected 1 h after WSSV injection and stained with DAPI (blue) to label cell nuclei. Scale bar = 15 μm. (B) The phagocytic rate of hemocytes calculated by the formula in Materials and methods. Five hundred hemocytes were counted under the fluorescence microscope in each experiment. Injection of dsGFP was used as the control. Eight shrimp were used in each group. (C) The phagocytic index of hemocytes calculated by the formula in Materials and methods. (D-F) Phagocytic analysis after overexpression of MjSRC. (D) Phagocytosis observed under the fluorescence microscope. (E) The phagocytic rate. (F) The phagocytic index. Trx-His tag mRNA was used as the control mRNA in the overexpression assays. (G) Phagocytosis was detected by flow cytometry. Intact hemocytes (R2), differentiated from virions and cell debris (R1), were analyzed only in this assay. (H and I) The phagocytic rate determined by flow cytometry after knockdown (H) or overexpression (I) of MjSRC. Five thousand hemocytes were counted in each assay. The data were statistically analyzed using Student’s t-test. *, p < 0.05.
Fig 4
Fig 4. MjSRC binds to the envelope protein of WSSV.
(A) Schematic representation of MjSRC indicating different domains (upper panel). Recombination and purification of GST-tagged rMjSRC-EX, rMjSRC-MAM, and rMjSRC-CCP, as well as Trx-His-tagged rVP19, rVP24, rVP26 and rVP28 (lower panel). The proteins were analyzed using SDS-PAGE and stained with Coomassie blue. (B) GST-pulldown assays to detect the interaction between rMjSRC-EX with rVP19, rVP24, rVP26 or rVP28. rMjSRC-EX could bind to rVP19 only. (C) GST-pulldown assays to detect the interaction between different domains of MjSRC, rMjSRC-MAM and rMjSRC-CCP, with rVP19. (D) The results of transmission electron microscopy. rMjSRC-EX was labeled with colloidal gold (10 nm), and then incubated with purified WSSV virions on carbon-coated nickel grids. After thorough washing, the grids were observed under a transmission electron microscope. The Trx-His tag was also labeled with colloidal gold and then incubated with virions as the control (I). (II) Intact virion, (III) nucleocapsid of WSSV. The arrow indicated colloidal gold. Scale bar = 500 nm. (E) Co-IP assays to confirm the interaction between MjSRC with VP19 in vivo. Anti-MjSRC and anti-VP19 serum were used to analyze the interaction in hemocytes derived from WSSV-infected shrimp. Normal rabbit IgG was used as the negative control. (F) The co-localization of MjSRC and FITC labeled-WSSV in shrimp hemocytes analyzed by immunocytochemistry. WSSV was labeled with FITC (green) and injected into shrimp. Hemocytes were collected at different time points (15, 30, 45 and 60 min) after WSSV injection. The primary antibody is anti-MjSRC and the second antibody is anti-rabbit IgG Alexa-546 (red). Nuclei were stained with DAPI (blue). Scale bar = 15 μm.
Fig 5
Fig 5. Oligomerization and internalization of MjSRC in hemocytes of shrimp infected by WSSV.
(A) Native PAGE of rMjSRC-EX. Renatured rMjSRC-EX (~ 60 KDa) and native protein marker (GE healthcare life science) were analyzed using native PAGE and stained with Coomassie blue. (B) A trimer of MjSRC was detected in vivo using western blotting after treatment of hemocytes with crosslinker (BS3). Shrimp were divided into two groups: injection with PBS or WSSV, respectively. After 30 min, hemocytes were collected from each group and treated with BS3. These hemocytes were homogenized and separated by SDS-PAGE. Western blotting was then performed using anti-MjSRC sera. β-Actin served as the reference. (C) Recombination and purification of GST-tagged rMjSRC-EX, rMjSRC-MAM, and rMjSRC-CCP, as well as His-tagged rMjSRC-EX, rMjSRC-MAM, and rMjSRC-CCP. These proteins were analyzed using SDS-PAGE and stained with Coomassie blue. (D) GST-pull-down assays to confirm the oligomerization of each domain of MjSRC. (E) Immunocytochemistry was performed using anti-MjSRC sera as the primary antibody. The secondary antibody was labeled with Alexa-488 (green). Cell nuclei were stained with DAPI (blue) and then observed under the fluorescence microscope. MjSRC were located on the surface of hemocytes in normal shrimp (without WSSV challenge) (upper panel) and were then gradually internalized into cytoplasm at 0.5 h and 1 h of WSSV challenge (middle and lower panel). Scale bar = 15 μm. (F) Subcellular distribution of MjSRC after WSSV challenge, as analyzed by western blotting. Proteins from the cytomembrane and cytoplasm of hemocytes were separated, respectively. Western blotting was performed using anti-MjSRC sera. β-Actin served as a marker of the cytoplasm.
Fig 6
Fig 6. Mjβ-arrestin2 interacts with MjSRC and is involved in the internalization of MjSRC in shrimp.
(A) GST-pull-down to analyze the interaction of the MjSRC extracellular region with Mjβ-arrestin 1 and 2. (B) GST-pull-down to analyze the interaction of the MjSRC intracellular region (MjSEC-IN) with Mjβ-arrestin 1 and 2. (C) GST-pull-down to analyze the interaction of MjSEC-IN with the N- or C-terminal domain of Mjβ-arrestin 2. (D) Co-IP was performed to detect the interaction between MjSRC with Mjβ-arrestin2 in shrimp using anti-MjSRC and anti-Mjβ-arrestin2 sera. (E) Shrimp were injected with dsGFP + WSSV, or dsMjβ-arrestin2 + WSSV, respectively. After 1 h, hemocytes were collected from each group, and immunocytochemistry was performed using anti-MjSRC sera as the primary antibody. The secondary antibody was labeled with Alexa-488 (green). Cell nuclei were stained with DAPI (blue) and then observed under the fluorescence microscope. Scale bar = 15 μm. (F) The percentage of cells showing MjSRC internalization in total detected cells. Two hundred cells were counted under the fluorescence microscope in each group. The experiment and cell counting were performed three times. (G) Subcellular distribution of MjSRC after knockdown of Mjβ-arrestin2 in WSSV-infected shrimp, as analyzed by western blotting. β-Actin was used as a marker of cytoplasmic proteins. (H) Efficiency of Mjβ-arrestin2 RNAi in hemocytes, as detected by qRT-PCR (upper panel) and western blotting (lower panel). (I) The quantification of viral copies from each group was detected by qRT-PCR. Significant differences were analyzed using Student’s t-test. *, p < 0.05. (J) The survival rate of Mjβ-arrestin2-RNAi shrimp infected with WSSV. After 24 h of dsRNA injection, WSSV inoculums were injected. Dead shrimp was monitored every day after WSSV injection. dsGFP injection was used as the control. The survival rate was calculated and the survival curves were presented as Kaplan-Meier plots. Differences between the two groups were analyzed with log-rank test using the software of GraphPad Prism 5.0. p = 0.02.
Fig 7
Fig 7. Hemocytes phagocytosis of WSSV is clathrin-dependent.
(A) Effect of chlorpromazine (CPZ) on the viability of shrimp. Shrimp were treated with increasing concentrations of CPZ for 2 days and the survival rate was calculated. (B) Shrimp were challenged with WSSV, and divided into four groups. After 24 h of WSSV challenge, different concentrations of CPZ were injected into each group, respectively. Another 24 h later, VP28 mRNA expression levels in the gills from each group were detected using RT-PCR (upper two panels) and the protein levels were analyzed using western blotting (lower two panels). (C) The amounts of WSSV DNA were compared between the two groups, using VP28 as a marker, and detected by qRT-PCR. The β-Actin gene served as the reference. (D) Expression patterns of Mjclathrin mRNA in hemocytes, stomach and intestine of shrimp after WSSV challenge, as detected by qRT-PCR. The β-Actin gene was used as the reference. Results were expressed as mean ± SD and analyzed statistically using Student’s t-test. *, p < 0.05. (E) Efficiency of Mjclathrin RNAi in hemocytes, as detected by qRT-PCR. (F) VP28 expression in Mjclathrin-RNAi shrimp were analyzed by western blotting. β-Actin served as the internal control. (G) The quantification of viral copies from each group was detected by qRT-PCR based on a standard curve. Shrimp were challenged with WSSV, 24 h later, dsGFP or dsMjclathrin RNA were injected into each group. The VP28 expression level or the viral copies were analyzed after 24 h of dsRNA injection. (H) Phagocytosis of hemocytes in dsMjclathrin knockdown shrimp observed under the fluorescence microscope. Shrimp were injected with dsGFP or dsMjclathrin. After 24 h, WSSV virions (labeled with FITC, green) were injected into each group. The hemocytes were collected 1 h after WSSV injection for observation. (h) Phagocytic rate was calculated based on the formula described in method. (I) The effect of inhibitor CPZ on phagocytic rate of hemocytes. After 1 h of CPZ injection, FITC-labeled WSSV were injected and 1 h later, hemocytes were collected. Cell nuclei were stained with DAPI (blue). (i) Phagocytic rate was calculated based on the formula described in method. Scale bar = 15 μm. Five hundred hemocytes were counted under the fluorescence microscope in each experiment. Student’s t test was used for statistic analysis. *, p < 0.05.
Fig 8
Fig 8. Lysosomes of hemocytes were involved in the clearance of WSSV in shrimp.
(A) Effect of chloroquine (CLQ) on the viability of shrimp. Shrimp were treated with increasing concentrations of CLQ for 2 days and the survival rate was calculated. (B) Shrimp were divided into four groups, 24 h post WSSV challenge, different concentrations of CLQ were injected into each group, respectively. Another 24 h later, VP28 mRNA expression levels in the gills from each group were detected using RT-PCR (upper two panels) and the protein levels were analyzed using western blotting (lower two panels). (C) VP28 mRNA expression levels in the gills from each group were detected by qRT-PCR. Results are expressed as mean ± SD and were analyzed statistically using Student’s t-test. **, p < 0.01. (D) Co-localization of ingested virions and lysosomes. After 24 h of dsMjβ-arrestin2 or dsMjclathrin injection, FITC-labeled WSSV virions were injected into shrimp. After another 1 h, hemocytes were collected, incubated with LysoBrite Red (BBI) to label lysosomes, stained with DAPI and then observed under a fluorescence microscope. Scale bar = 15 μm. dsGFP injection was used as the control. (E) Statistical analysis of the co-localization between WSSV and lysosomes. The amounts of co-localized WSSV with lysosomes were counted under the fluorescence microscope in each group. The rate of co-localization of WSSV and lysosome in dsMjβ-arrestin2- or dsMjclathrin-RNAi group was normalized to the control group. Student’s t-test was used for statistic analysis. *, p < 0.05.
Fig 9
Fig 9. Schematic representation of the antiviral mechanism of MjSRC.
MjSRC locates on the cell membrane of hemocytes, with two CCP domains and one MAM domain projected out of the cell. After WSSV infection, MjSRC oligomerized to a trimer via its CCP and MAM domains. Subsequently, the intracellular region of MjSRC recruits and interacts with an adaptor protein, Mjβ-arrestin2, and the latter interacts with Mjclathrin to induce internalization of MjSRC with WSSV via a clathrin-coated vesicle. This vesicle that holds the foreign pathogens (WSSV) is called a phagosome, which becomes a phagolysosome after fusing with a lysosome. Ultimately, WSSV is eliminated by various enzymes.

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References

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This work was supported by grants from National Natural Science Foundation of China (Grants 31630084, 31130056 and 31472303) (http://www.nsfc.gov.cn/publish/portal1/) and National Basic Research Program of China (No. 2012CB114405) (http://www.most.gov.cn/eng/programmes1/200610/t20061009_36223.htm). JXW received the fundings. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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