Exosome-mediated viral nucleic acid presentation in a crustacean reveals innate immunity evolution from a novel perspective

As an enduring hot topic in the field of innate immunity, apoptosis is widely considered as an effective approach to eliminate pathogenic microbes, and possesses crucial role during host-pathogen interactions. Recently, researchers have found that virus-containing host cells could transmit apoptotic signals to surrounding uninfected cells during infection, but the mechanism remains unclear. Here, we found that exosomes secreted by WSSV-infected mud crab hemocytes contain viral nucleic acid wsv277, which could be transported to the recipient cells and further expressed viral protein with phosphokinase activity. Besides, by using transcriptome, proteome, ChIP-seq and coIP techniques, the results revealed that wsv277 could activate the transcription and translation of apoptotic genes via interacting with CBF and EF-1α, so as to suppress the spread of virus infection by inducing apoptosis of the surrounding cells. Therefore, for the first time, our study proved that the components of DNA virus could be encapsulated into exosomes, and elucidated the mechanism of apoptotic signal transduction between cells from the perspective of exosome. Moreover, comparing those of invertebrates, we hypothesized that the innate immune system of vertebrate was weakened or degenerated, allowing exosomes to be employed by virus as the vehicle for immune escape. Significance statement Our study revealed that the components of DNA virus could be packaged and transmitted through the exosomes of lower invertebrates, which strongly demonstrated the diversity of exosome-mediated viral immunity and its universality in animals. Furthermore, we elucidated the mechanism of apoptotic signal transduction between cells from the perspective of exosome, and revealed a novel strategy for the host to cope with viral infection. Since exosome is an important innate immune regulation approach, we hypothesize that the innate immune system of vertebrates is weakened or degenerated, allowing exosomes to be employed by virus as the vehicle for immune escape.


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As an enduring hot topic in the field of innate immunity, apoptosis is widely 24 considered as an effective approach to eliminate pathogenic microbes, and possesses 25 crucial role during host-pathogen interactions. Recently, researchers have found that 26 virus-containing host cells could transmit apoptotic signals to surrounding uninfected 27 cells during infection, but the mechanism remains unclear. Here, we found that  In an attempt to comprehensively reveal the mechanism of exosomes in regulating 105 host immune response and the impact on the fate of viral infection process, WSSV 106 (White spot syndrome virus)-infected mud crab Scylla paramamosain was used as the 107 research model in this study. Herein, we found that five viral mRNAs were specifically 108 packaged by exosomes during the infection process. Among them, wsv277 could be 109 delivered to surrounding exosome-receiving cells and translated into viral proteins, and 110 further mediated the transcription and translation of apoptotic genes through interacting 111 with CBF (CCAAT-binding factor) and EF-1α (Elongation factor 1 alpha), which 112 eventually suppressed virus invasion by inducing apoptosis of neighboring cells.

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Screening of viral nucleic acids in the exosomes of WSSV-infected mud crab 116 In order to determine whether exosomes secreted by invertebrate are capable of 117 transporting viral genomic nucleic acids like mammals, we tried to analyze the nucleic 118 acid composition in the exosomes of mud crabs before/ after WSSV infection. The 119 typical cup-shaped structure of exosomes collected from the hemolymph of WSSV-120 injected (exosome-WSSV) and PBS-injected (exosome-PBS) mud crabs were observed 121 under TEM (Transmission electron microscopy) (Fig. 1A). Besides, the quantities and 122 sizes of the isolated exosomes were measured by nanoparticle tracking analysis (NTA) 123 (Fig. 1B), and the purity were further ascertained via detecting the exosomal markers 124 CD63, CD9, TSG101 and the cytoplasmic marker Calnexin (Fig. 1C). The data indicate 125 successful isolation of exosomes from mud crab challenged with WSSV or PBS. 126 Next, transcriptome sequencing was performed on the above isolated exosomes 127 (Fig. 1D), and 5 WSSV-specific nucleic acids (wsv001, wsv091, wsv271, wsv277 and 128 wsv447) were identified in exosome-WSSV group via blasting with WSSV genome 129 (Fig. 1E). Then, PCR and qPCR were both conducted to confirm the existence of the 130 viral nucleic acids ( Fig. 1F and 1G). Taken together, the above findings revealed that 5 131 viral mRNAs were specific encapsulated in the exosomes of mud crab during WSSV 132 infection, and the level of wsv277 was the highest among them. 133 wsv277-containing exosomes suppress WSSV replication via apoptosis 134 Our previous study showed that exosome secreted from WSSV-challenged mud 135 crab could inhibit virus infection by inducing apoptosis 19 , based on this consideration, 136 we tried to explore whether this process was mediated by the viral mRNAs packaged    Furthermore, through western blot and immunofluorescence analysis, it was found that 159 wsv277 mRNA packaged by exosomes could be translated into viral protein in 160 hemocytes ( Fig. 3C and 3D). 161 Then, to reveal the potential functions of the translated wsv277 protein, the amino 162 acid sequence of wsv277 was subjected to domain prediction by bioinformatics tools, 163 and phosphorus residues were found on PHE405, PHE418, PHE420 and ASN422 in 164 the tertiary structure (Fig. 3E), indicating that wsv277 protein may possess 165 phosphokinase activity. To confirm this conjecture, we detected the phosphorylation 166 ability of rwsv277 (recombinant wsv277 protein) to hemocyte lysates, and found that 167 rwsv277 was capable to phosphorylate hemocyte lysates in vitro (Fig. 3E). In addition, 168 we co-injected exosomes and wsv277-siRNAs into mud crabs and then measured the  Screening and validation of wsv277-interacting proteins 176 Kinases are generally considered to be tightly relevant to the regulation of gene 177 expression 27 , for this reason, we performed transcriptome analysis on the hemocytes 178 of mud crab challenged with exosomes and wsv277-siRNA (Fig. 4A). Among the 179 differentially expressed genes, 428 genes were found to be altered in Exo-WSSV group 180 compared with Exo-PBS, and with no significant difference when wsv277 was silenced 181 (Fig. S2A), indicating that wsv277 was required for the transcription of these genes.

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Besides, KEGG and GO analysis showed that many of wsv277-regulated genes were 183 involved in apoptosis process ( Fig. 4B and S2B), which was consistent with the above 184 data. Next, combined with GST pull-down and mass spectrometry, we obtained a series 185 of potential wsv277-interacting proteins in mud crab ( Fig. 4C and 4D). Among them, 186 transcription factor CBF and translation elongation factor EF-1α were selected since for CBF and EF-1α were co-injected into WSSV-challenged mud crabs, and the data of 205 annexin V, caspase 3 activity and mitochondrial membrane potential analysis showed 206 that silencing of CBF and EF-1α remarkably decreased apoptosis levels compared with 207 control group (Fig. 5F-5H). In addition, through detecting the copy number of virus 208 particles, we found that CBF and EF-1α were required for exosome-mediated WSSV 209 suppression (Fig. 5I). Taken together, these finding suggested that CBF and EF-1α were 210 the downstream targets for exosome-induced apoptosis and subsequent suppression of 211 viral replication.

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Underlying mechanisms of wsv277-CBF complex-mediated apoptosis 213 To reveal the mechanism and role of CBF in exosome-mediated apoptosis, we 214 tried to analyze the phosphorylation levels of CBF after exosomes treatment, since the 215 above findings indicated that wsv277 protein possess phosphokinase activity. The 216 results showed that Exo-WSSV could phosphorylate CBF at tyrosine sites by packaging 217 wsv277 (Fig. 6A). Then, the influence of the phosphorylation modification on the 218 cellular localization of CBF was detected, the results of both western blot analysis and 219 immune-fluorescence detection revealed that CBF would be translocated to the cell 220 nucleus after phosphorylation by wsv277 ( Fig. 6B and S4).

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Next, we analyzed the downstream target genes of transcription factor CBF by 222 ChIP-seq using anti-CBF IgG (Fig. 6C). The sequencing data of ChIP products showed 223 that the reads distribution across genomic regions of CBF possess certain specificity 224 (Fig. 6D), and the promoter sequences bound with CBF protein could be classified into 225 3 motifs (Fig. 6E). Then, KEGG analysis conducted on these genes revealed that many 226 of CBF-regulated genes were involved in apoptosis pathway (Fig. 6F). To confirm these 227 findings, five of CBF-regulated genes that have been reported to mediate apoptosis 228 process were selected for further validation. The results showed that Exo-WSSV could 229 promote the transcription of these apoptotic genes, and the positive effects were 230 significantly inhibited when wsv277 or CBF was silenced (Fig. 6G). Taken together, 231 the above data suggested that exosomes could mediate phosphorylation and nuclear 232 translocation of CBF by wsv277, and then promote the transcription of apoptotic genes.  The next issue is to identify the downstream proteins synthesized by EF-1α, thus, 248 proteomic analysis was performed in the hemocytes after EF-1α knockdown. The most 249 significantly downregulated proteins were subjected to KEGG analysis, and the results 250 revealed that many of EF-1α-synthesized proteins were relevant to apoptosis pathway 251 ( Fig. 7G and 7H). To confirm the findings, five EF-1α-synthesized proteins that have 252 been reported to mediate apoptosis process were selected for further validation, the 253 results showed that Exo-WSSV could accelerate the synthesis of apoptotic proteins, 254 and the acceleration effects was reduced after wsv277 or EF-1α silencing (Fig. 7I). 255 Taken together, the above data suggested that wsv277 packaged by exosomes could 256 cooperated with EF-1α, so as to accelerate the synthesis of apoptotic proteins.

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In summary, the findings in this study indicated that during WSSV infection, viral   (Fig. 8B). Therefore, since exosome 327 is an important innate immune regulation approach, we hypothesize that the innate 328 immune system of vertebrates is weakened or degenerated, allowing exosomes to be 329 employed by virus as the vehicle for immune escape.

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Ethics statement 332 The mud crabs used were taken from Niutianyang aquaculture farm (Shantou, 333 China). No specific permits were required since mud crab was not belonged to 334 endangered or protected species. The animals were processed according to "the          antibody-resistant spread of classical swine fever virus in cell culture.