key: cord-0754227-gs5xy5zd
authors: Su, Hang; Liao, Zhiwei; Yang, Chunrong; Zhang, Yongan; Su, Jianguo
title: Grass Carp Reovirus VP56 Allies VP4, Recruits, Blocks, and Degrades RIG-I to More Effectively Attenuate IFN Responses and Facilitate Viral Evasion
date: 2021-09-15
journal: Microbiology spectrum
DOI: 10.1128/spectrum.01000-21
sha: c3db7a8ce230fbf987adfa9641a5abeb87529c30
doc_id: 754227
cord_uid: gs5xy5zd

Grass carp reovirus (GCRV), the most virulent aquareovirus, causes epidemic hemorrhagic disease and tremendous economic loss in freshwater aquaculture industry. VP56, a putative fibrin inlaying the outer surface of GCRV-II and GCRV-III, is involved in cell attachment. In the present study, we found that VP56 localizes at the early endosome, lysosome, and endoplasmic reticulum, recruits the cytoplasmic viral RNA sensor retinoic acid-inducible gene I (RIG-I) and binds to it. The interaction between VP56 and RIG-I was detected by endogenous coimmunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was then confirmed by traditional co-IPs and a novel far-red mNeptune-based bimolecular fluorescence complementation system. VP56 binds to the helicase domain of RIG-I. VP56 enhances K48-linked ubiquitination of RIG-I to degrade it by the proteasomal pathway. Thus, VP56 impedes the initial immune function of RIG-I by dual mechanisms (blockade and degradation) and attenuates signaling from RIG-I recognizing viral RNA, subsequently weakening downstream signaling transduction and interferon (IFN) responses. Accordingly, host antiviral effectors are reduced, and cytopathic effects are increased. These findings were corroborated by RNA sequencing (RNA-seq) and VP56 knockdown. Finally, we found that VP56 and the major outer capsid protein VP4 bind together in the cytosol to enhance the degradation of RIG-I and more efficiently facilitate viral replication. Collectively, the results indicated that VP56 allies VP4, recruits, blocks, and degrades RIG-I, thereby attenuating IFNs and antiviral effectors to facilitate viral evasion more effectively. This study reveals a virus attacking target and an escaping strategy from host antiviral immunity for GCRV and will help understand mechanisms of infection of reoviruses. IMPORTANCE Grass carp reovirus (GCRV) fibrin VP56 and major outer capsid protein VP4 inlay and locate on the outer surface of GCRV-II and GCRV-III, which causes tremendous loss in grass carp and black carp industries. Fibrin is involved in cell attachment and plays an important role in reovirus infection. The present study identified the interaction proteins of VP56 and found that VP56 and VP4 bind to the different domains of the viral RNA sensor retinoic acid-inducible gene I (RIG-I) in grass carp to block RIG-I sensing of viral RNA and induce RIG-I degradation by the proteasomal pathway to attenuate signaling transduction, thereby suppressing interferons (IFNs) and antiviral effectors, facilitating viral replication. VP56 and VP4 bind together in the cytosol to more efficiently facilitate viral evasion. This study reveals a virus attacking a target and an escaping strategy from host antiviral immunity for GCRV and will be helpful in understanding the mechanisms of infection of reoviruses.

proteasomal pathway to inhibit RIG-I-regulated IFN antiviral responses, resulting in GCRV replication and infection enhancement and viral evasion. In addition, VP56 binds to the GCRV major outer capsid protein VP4 in the cytosol, synergistically enhancing the degradation of RIG-I and more efficiently facilitating viral evasion. The present results reveal a viral invasion target and viral evasion strategy developed by aquareovirus involving the negative regulation of RIG-I. This study provides insights into the functions of viral fibrin and the viral escaping strategy and infection mechanisms of reoviruses.

VP56 binds to the helicase domain of RIG-I. To research the molecular action of VP56 during GCRV infection, endogenous coimmunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) were performed (Fig. 1) . Recombinant GST-VP56 fusion protein was expressed in Escherichia coli BL21(DE3) pLysS cells and purified by affinity chromatography (Fig. 1A) . The polyclonal antibodies (Abs) of GST-VP56 and GST were prepared by inoculating New Zealand White rabbits and were purified by antigen-antibody affinity chromatography, respectively. For endogenous co-IP, C. idella kidney (CIK) cells were infected with GCRV, and a co-IP was performed with VP56 polyclonal Ab, GST Ab, and negative serum, respectively, followed by LC-MS/MS (Fig. 1B, left) . For GST pulldown, CIK cell proteins were extracted and incubated with VP56 protein for binding, while GST protein was used as a control. GST resin was then added into the complex. After washing and elution, the eluted protein mixture was analyzed by LC-MS/MS (Fig. 1B, right) . Summarizing the LC-MS/MS results, several interactive proteins were identified from the proteomic data set, of which 77 candidate proteins matched at least two peptide fragments in a protein sequence (Table S1 in the supplemental material). Among the candidate interactive proteins, RIG-I was noticed.

To understand the molecular action of fibrin VP56 during GCRV infection, subcellular localization of VP56 was detected by confocal microscopy (Fig. S1 ). Fathead minnow (FHM) cells were cotransfected with VP56-green fluorescent protein (GFP) fusion vector and relevant organelle protein markers followed by 49,6-diamidino-2-phenylindole (DAPI) staining for the cell nucleus. As shown in Fig. S1 , appearances of yellow signals indicating overlapping of green and red were observed in cells cotransfected with VP56 and RAB5, LAMP2, and GRP78, which are the marker proteins of early the endosome, lysosome, and endoplasmic reticulum (ER), respectively. These results demonstrated that VP56 localizes at the early endosome (Fig. S1A) , lysosome (Fig. S1C) , and ER ( Fig. S1D ) but not the late endosome (Fig. S1B ). Colocalizations of VP56 with cytoplasmic organelles imply the possible distribution of VP56 after virus uncoating in the cytoplasm.

The direct interaction between VP56 and RIG-I was further confirmed by traditional co-IPs ( Fig. 2A) and far-red mNeptune-based bimolecular fluorescence complementation (BiFC) (Fig. 2B ). Positive bands were observed in both co-IP results conducted by RIG-I pulling down of VP56 and VP56 pulling down of RIG-I via tag Abs ( Fig. 2A) . According to the BiFC results, red fluorescence of mNeptune was not detected in single VP56 or RIG-I proteins overexpressed in cells. When CIK cells were transfected with both VP56 and RIG-I plasmids linked with each terminal of the mNeptune protein, these two fusion proteins present closely and send out red fluorescence signal (Fig. 2B) . To further find out the specific interaction domain of RIG-I, a co-IP was performed, and the results indicated that the RIG-I helicase domain could interact with VP56 (Fig. 2C) . Therefore, the interaction between VP56 and RIG-I was proven, and the binding site was found to be the helicase domain of RIG-I.

VP56 enhances K48-linked ubiquitination to degrade RIG-I by the proteasomal pathway. Ubiquitination modifications of RIG-I play a pivotal role in the activation and attenuation of innate antiviral immunity. The effects of VP56 on expression and ubiquitination of RIG-I protein were investigated (Fig. 3) . Protein expression of RIG-I decreased along with increasing VP56 (Fig. 3A) , indicating that VP56 degrades RIG-I. MG132, a proteasomal pathway inhibitor, was then used to test whether VP56 degrades RIG-I via the proteasome pathway. The results demonstrated that MG132 rescues the inhibited RIG-I production by VP56, suggesting that VP56 degrades RIG-I through the proteasome pathway (Fig. 3B) . Furthermore, ubiquitination assays were Co-IP was performed with anti-Flag monoclonal Ab and mouse IgG (control) and immunoblotting with the corresponding Abs. IB, immunoblot; WCL, whole-cell lysate. (B) Imaging of the VP56-RIG-I interaction by far-red mNeptune-based BiFC. Corresponding vectors were transfected alone or cotransfected into CIK cells under normal conditions. In the BiFC system, the fluorescence of the mNeptune channel was red, and the nucleus was stained with DAPI. Images were acquired using confocal microscopy under a 40Â lens objective. Appearance of red fluorescence represents the positive observation. BiFC experiments were repeated in triplicate, and the images were selected and cropped to show the positive results. (C) VP56 interacts with the RIG-I-DExD/H helicase domain. FHM cells were cotransfected with 4 mg of VP56-GFP and 4 mg of RIG-I-CARD-HA or RIG-I-helicase-HA or RIG-I-RD-HA for 24 h in 10-cm 2 dishes. Co-IP was performed using HA Ab, and mouse IgG was used as a control. IPs were analyzed by immunoblotting with anti-HA and anti-GFP, respectively. Expression of VP56-GFP (input) was examined with GFP Ab. All the co-IP and BiFC assays were repeated independently at least three times. performed in FHM cells ( Fig. 3C and D) . These results showed that VP56 enhances ubiquitination of RIG-I (Fig. 3C ). When K63-and K48-linked ubiquitination were measured, respectively, the results showed that VP56 enhances both K63-and K48-linked ubiquitination of RIG-I in a dose-dependent manner (Fig. 3D) . Collectively, VP56 degrades RIG-I via K48-linked ubiquitination for the proteasomal pathway, and the immune-activated functions of K63-linked ubiquitination increased due to VP56 might be counteracted by RIG-I protein degradation because of VP56.

VP56 inhibits RIG-I-triggered IFNs and inflammation responses. RIG-I is a crucial PRR in the antiviral signaling pathway, sensing viral dsRNA in the cytoplasm. VP56 binding and degrading of RIG-I is supposed to be beneficial for GCRV evasion and replication. At the promoter level, VP56 suppresses the promoter activities of the key molecules in the RLR signaling pathway, including RIG-I, IPS-1, STING, TBK1, and IRF3 by dual-luciferase assays (Fig. 4A) . At the mRNA level, a similar tendency was observed by quantitative real-time RT-PCR (qRT-PCR) (Fig. 4B) . Furthermore, IRF3 protein and phosphorylation levels were examined by Western blotting (WB). The results indicated that VP56 attenuates the protein expression and phosphorylation of IRF3 (Fig. 4C) , which implied that the transcription of antiviral immune molecules would be reduced. Therefore, we further investigated the influence of VP56 on representative IFNs and inflammation factors at the promoter and transcription levels. The results indicated that VP56 reduces the promoter activities and mRNA expression of IFN1, IFN3, IFN-g2, and NF-k B1 ( Fig. 5A and Rel. Luc. Act., relative luciferase activity. (B) VP56 decreases RLR-related gene mRNA expression in uninfected (top) or GCRV-infected (bottom) CIK cells. CIK cells transiently transfected with VP56/empty vector were seeded in 12-well plates. After 24 h, CIK cells were uninfected or infected with GCRV. Twenty-four hours postinfection, total RNAs were extracted, and mRNA expression was examined for RIG-I, IPS-1, STING, TBK1, and IRF3 genes. Data of reporter assays and qRT-PCR are shown as mean 6 standard deviation (SD) of 4 wells of cells per group with three independent experiments. Significance was calculated in relation to the control group; *, P , 0.05; **, P , 0.01. The relative transcription levels were normalized to mRNA expression of the EF1a gene and are represented as fold change relative to the transcription level in control cells, which was set to 1. (C) VP56 suppresses IRF3 protein and phosphorylation levels. CIK cells transiently transfected with VP56/empty vector were seeded in 6-well plates. After 24 h, cell lysate was used for Western blotting using IRF3 polyclonal antiserum. b-Tubulin was used to normalize the protein concentration. All the experiments were repeated independently at least three times. The histograms beside the Western blotting results show the IRF3 and phospho-IRF3 (IRF3-P) expression levels, which were quantified using ImageJ software. B). Moreover, VP56 weakens the protein expression of IFN1 and NF-k B1 (Fig. 5C ). In addition, when VP56 was cotransfected with RIG-I, the promoter activities and mRNA levels of IFN1, IFN3, IFN-g2, and NF-k B1 were suppressed by VP56 compared with RIG-I alone ( Fig. 5D and E), indicating that VP56 inhibits the RIG-I-triggered IFN and inflammatory responses.

VP56 suppresses antiviral effectors and boosts GCRV evasion. To determine whether VP56 interferes with antiviral effectors downstream of the IFN pathway to facilitate viral evasion, we examined mRNA expression of representative IFN-stimulated genes (ISGs) (myxovirus-resistant 2 [Mx2] and GCRV-induced gene 1 [gig1]), viral genes (VP1, VP4, NS38, and VP35), viral titers (50% tissue culture infective dose [TCID 50 ]), and cell death caused by VP56 (Fig. 6 ). mRNA expression of antiviral effectors Mx2 and gig1 were reduced by VP56 (Fig. 6A) . After GCRV infection, viral VP1, VP4, NS38, and VP35, which are encoded by GCRV-II segment S1, S6, S10, and S11, respectively, were increased by VP56 (Fig. 6B ). Furthermore, a TCID 50 assay indicated that VP56 increases the titer of GCRV in CIK cells when transiently or stably transfecting VP56 (Fig. 6C ). In addition, crystal violet staining also illustrated that VP56 promotes cell death caused by GCRV infection (Fig. 6D ). All the results demonstrated that VP56 inhibits ISG expression and facilitates viral RNA synthesis, leading to increased GCRV titer and cytopathic effect.

Confirmation of VP56-dependent repression of the RIG-I-triggered antiviral immune pathway. To further address the regulation mechanism of VP56 on the host antiviral signaling pathway, RNA sequencing (RNA-seq) was performed with VP56/ empty vector stable expression in CIK cells (Fig. 7) . KEGG functional enrichment of differentially expressed (fold change of at least 4) genes (DEGs) analysis showed that pathways in cancer, FoxO signaling, and extracellular matrix (ECM)-receptor interaction were activated the most (Fig. 7A) , showing that these three signaling pathways have the strongest responses after VP56 stimulation. Raw expression levels of the RIG-Irelated signaling pathway are shown in Fig. 7B , and representative genes (RIG-I, IPS-1, STING, TBK1, IRF3, IFN1, IFN3, IFN-g2, NF-k B1, and Ik Ba) were selected for validation by qRT-PCR. The results showed that the RLR antiviral signaling pathway and inflammation factor NF-k B were inhibited by VP56 (Fig. 7C to E); meanwhile, expression of Ik Ba, an NF-k B suppressor gene, was enhanced (Fig. 7E) .

VP56 knockdown potentiates IFN responses and reduces GCRV replication and titer (Fig. 8) . The CIK cells that were stably transfected with VP56 were used to investigate the influence of VP56 knockdown on host antiviral immune genes and viral replication. si-VP56-1 displayed the highest interference efficiency among the three small interfering RNAs (siRNAs) by qRT-PCR (Fig. 8A ). VP56 knockdown increases the representative IFN (IFN1, IFN3, and IFN-g2) and inflammation factor (NF-k B1) responses (Fig. 8B) . These results were further confirmed at the protein level (Fig. 8C) . Furthermore, VP56 knockdown inhibits GCRV replication and titer ( Fig. 8D and E) . All the results indicated that VP56 knockdown enhances antiviral immunity and inhibits GCRV replication.

VP56 allies VP4, synergistically facilitating viral evasion. In our previous study, the GCRV major outer capsid protein VP4 binds to the CARD and RD domains of RIG-I, degrades RIG-I by the proteasomal pathway, and promotes viral replication (29) . We therefore wondered whether there is some interaction between VP56 and VP4. We investigated the interaction and joint effect between VP56 and VP4 (Fig. 9 ). When VP4-GFP and VP56-red fluorescent protein (RFP) were cotransfected in FHM cells, we found that VP56 colocalizes with VP4 (Fig. 9A) . Furthermore, co-IPs with VP56 pulling down VP4 and VP4 pulling down VP56 via different fusion tag Abs were performed, and the results demonstrated that VP56 and V4 bind together in the cytosol (Fig. 9B ). After that, we cotransfected VP56 and VP4 and found that VP56 and VP4 synergistically degrade RIG-I (Fig. 9C) , and mRNA expression of representative antiviral immune genes IFN1 and Mx2 were inhibited strongly (Fig. 9D) ; meanwhile, the expression of representative viral genes VP1 and VP35 was increased intensively (Fig. 9E) . Titers of GCRV were increased by VP56 and VP4 synergistically (Fig. 9F) . The results indicated that surface fibrin VP56 and major outer capsid protein VP4 of GCRV bind together and synergistically degrade RIG-I, suppress antiviral immune responses, and accomplish GCRV immune evasion.

VP56 encoded by segment 7 is a fiber protein on the outer capsid of the GCRV-II/III particle (9) , which is involved in cell attachment (10) . VP56 is similar to the s 1 protein of mammalian reovirus (MRV) (8, 30) . In mammals, junctional adhesion molecule A (JAM-A) is the only known cell surface receptor for MRV (31) , but the function of JAM-A in fish is not certain (32) . Several attempts have been made to find out the membrane receptor of aquareovirus, including fibulin-4, Ubc9, LITAF, LamR, TIA1, etc. (33) (34) (35) (36) (37) . However, the receptor of GCRV-II on the cell surface is still not determined. Further research investigating GCRV fibrin may provide insight into GCRV infection mechanisms.

RIG-I is a crucial PRR recognizing viral RNA in the cytoplasm. Interaction between VP56 and RIG-I was found by endogenous co-IP, GST pulldown, and subsequent LC-MS/MS (Fig. 1B) . All methods detected the binding. Subsequently, we further verified the interaction through traditional forward and reverse co-IPs ( Fig. 2A) and a far-red mNeptune-based bimolecular fluorescence complementation system (Fig. 2B) , which is a novel visible technique to research molecular interaction (38) . The helicase domain of RIG-I is critical for recognition of RNA, binding to the sugar-phosphate backbone of duplexed RNA, which results in release of CARDs (39) . The location of the binding site in the helicase domain (Fig. 2C) implies that VP56 may interfere with RIG-I binding dsRNA, which may affect the RIG-I-mediated antiviral signaling pathway.

It is widely believed that many host cellular physiologic processes are regulated by the ubiquitin system. It is of great importance in protein stability, immune activation, and host-pathogen interactions (12) . In mammals, RIG-I posttranslational modification by K63-linked or K48-linked ubiquitination is important for immune regulation functions (40) . In fishes, K63-or K48-linked RIG-I ubiquitination shows similar functions in mammals (22, 29, 41) . Tripartite motif containing 25 (TRIM25) and Riplet mediate K63linked ubiquitination in residues in RIG-I-CARDs for downstream IPS-1 recruitment and signal transduction (42) . The K48-conjugated ubiquitination chain is mediated by RNF125 to deliver substrates to proteasomes for degradation. This process guarantees a basal protein level for subsequent rapid signal activation (43) . GCRV-II VP56 induces both K63-and K48-linked ubiquitination in the present assays (Fig. 3) . The K48-linked proteasomal degradation pathway induced by VP56 plays a stronger role in the effect on RIG-I, leading to reduced downstream IFNs and inflammatory responses ( Fig. 4 and  5) . These results demonstrate that VP56 aims at and degrades RIG-I to weaken downstream signaling transduction via posttranslation modification.

RIG-I senses viral RNA, which triggers downstream cascade signals through IPS-1, STING, TBK1, and IRFs to promote IFN production for antiviral responses (44) . Independent of GCRV infection, VP56 inhibits RIG-I and downstream signaling molecules at promoter and mRNA levels ( Fig. 4A and B) . Downstream IRF3 was obviously suppressed by VP56 at protein and phosphorylation levels in a dose-dependent manner (Fig. 4C) . Correspondingly, IFNs were reduced by VP56 at mRNA and protein levels (Fig. 5) . As a result, antiviral effectors were repressed, and GCRV accomplished immune evasion (Fig. 6) . Additionally, when cotransfected with RIG-I, VP56 blocks RIG-I-triggered IFNs at promoter and mRNA levels, suggesting that VP56 targets RIG-I ( Fig. 5D and E) . VP56 also reduces NF-k B expression (Fig. 5) , whose translocation from the cytoplasm to the nucleus induces the expression of proinflammatory cytokines. These results were further confirmed by RNA-seq (Fig. 7) and siRNA experiments (Fig. 8) . The present study revealed that fibrin VP56 targets and degrades the RNA sensor RIG-I, thereby attenuating the signaling pathway and antiviral effectors, boosting viral immune evasion.

The IFN response is of great significance in host innate immunity against both viral and bacterial infections (45) . The RLR-mediated IFN signaling pathway is an important target for viral antagonism or escape to facilitate viral infection (46, 47) . Herpes simplex virus 1 (HSV-1) tegument protein UL37 targets the helicase domain of RIG-I to deamidate RIG-I, inhibiting RNA-induced activation of innate immune signaling (28) . STING is an important molecule in the RLR-IFN pathway and is often targeted by viruses. Hepatitis C virus (HCV) NS4B, a nonstructural protein, interacts with STING protein to restrain IFN responses (48) . NS2B3, a protease of dengue virus (DENV), inhibits IFN production via cutting STING (49, 50) . Papain-like proteases (PLP) of human coronavirus (HCoV) NL63 and severe acute respiratory syndrome coronavirus (SARS-CoV) inhibit IRF3 activation by destroying the STING dimer, thereby antagonizing innate immune signal transduction (51) . TBK1 is another important target for virus antagonism in the RLR-IFN pathway (52) . PLP2 of mouse hepatitis virus A59 (MHV-A59) affects polyubiquitination modification of TBK1 to block its kinase activity (53) . NS3 and NS2 proteins of HCV physically interact with TBK1 to competitively inhibit activation of downstream IRF3 (54) . IRF3/7 are crucial IFN regulation factors. In a previous report, GCRV VP56 represses IFN production by degrading phosphorylated IRF7 (11) . In the present study, we found that VP56 targets and degrades the immune initial point molecule RIG-I Fig. 9C , and the total RNAs were prepared for qRT-PCR assays of IFN1 and Mx2 genes. (E) VP56 unites VP4 to more efficiently facilitate viral replication. FHM cells were cotransfected, and gene expression (VP1 and VP35) was quantified as in Fig. 9D . (F) VP56 and VP4 promote GCRV infection. CIK cells transfected with empty vector/VP4/VP56/VP41VP56 were seeded in 6-well plates overnight and infected with GCRV, and the supernatants were collected at 24 h postinfection for viral titer assays by TCID 50 . All the experiments were performed in triplicate. (Fig. 3A) and boosts viral evasion (Fig. 6B) ; meanwhile, it also decreases protein and phosphorylation levels of IRF3 (Fig. 4C) .

Major outer capsid protein VP4 of GCRV has been reported to localize at the early endosome, lysosome, and endoplasmic reticulum (ER) but not the late endosome, which is the same location as VP56 (29) . VP4 also binds to RIG-I but at different domains (CARDs and RD) and promotes K48-linked ubiquitination of RIG-I to degrade it, resulting in low IFN responses (29) . VP56 and VP4 bind together in the cytosol, localize at the early endosome, lysosome, and ER, recruit and wrap the three domains of RIG-I, degrade RIG-I by the proteasomal pathway, and unite to synergistically suppress antiviral immunity and more effectively facilitate viral replication; this demonstrates that two viral outer proteins cooperatively aim at the initial point molecule of immune responses, RIG-I, in the process of GCRV infection, synergistically inhibiting antiviral immunity and more efficiently accomplishing viral immune evasion.

The present work reveals that GCRV fibrin VP56 acts as a forceful weapon for GCRV infection to inhibit IFN responses and boost viral evasion (Fig. 10) . GCRV invades cells, the virus uncoats actively or passively, and outer fibrin VP56 is released and disperses at the early endosome, lysosome, ER, etc., where VP56 and VP4 bind together, recruit and wrap the different regions (helicase domain for VP56, CARDs and RD for VP4) of the viral RNA sensor RIG-I (one of the initial point molecules of antiviral immunity), block and prevent RIG-I from sensing viral RNA, and, furthermore, facilitate K48-linked ubiquitination of RIG-I to degrade it via the proteasomal pathway, thus the initial immune function of RIG-I is impeded by dual mechanisms (blockade and degradation). The inhibited RIG-I signal leads to restrained IFN antiviral responses and enhanced GCRV replication and infection. VP56 cooperates with VP4 to synergistically reduce host antiviral immunity during GCRV infection to more effectively accomplish viral immune evasion. These results clarify the functions and mechanisms of VP56 in GCRV infection and reveal an escaping strategy of RNA virus, providing insight into the mechanisms of viral evasion and antiviral immunity.

Cells. CIK (C. idella kidney) and FHM (fathead minnow) cells were respectively cultured in Dulbecco's modified Eagle's medium (DMEM) and M199 supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin (Sigma), and 100 mg/ml of streptomycin (Sigma). Cells were incubated at 28°C with 5% CO 2 humidified atmosphere. Stably expressed CIK cell lines were screened with G418.

Virus and infection. The GCRV-II GCRV-097 strain was conserved in our lab. For viral infection, CIK cells were plated for 24 h in advance and then infected with GCRV-097 at a multiplicity of infection (MOI) of 1 as previously described (41) . TCID 50 assay. Samples were infected with the GCRV-097 strain (MOI = 1) for 24 h. The supernatants were serially diluted 10-fold and incubated with CIK cells in a flat 96-well plate to determine the 50% tissue culture infective dose (TCID 50 ). Cells were incubated at 28°C for 7 days. On day 7, the plates were examined for the presence of viral cytopathic effect under the microscope.

Antibodies. Mouse IFN1 polyclonal antibody (Ab) was prepared and conserved by our lab. Anti-IRF3 rabbit polyclonal antiserum was previously prepared and presented by Yibing Zhang, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China (25) . These Abs were tested by Western blotting before experiments. The VP56 Ab was produced and conserved in our lab. Hemagglutinin (HA) tag (ab18181) mouse monoclonal Ab, Flag tag (ab125243) mouse monoclonal Ab, and b-tubulin rabbit polyclonal Ab (ab6046) were purchased from Abcam. GFP mouse monoclonal Ab (AE012) and GST mouse monoclonal Ab (AE001) were purchased from Abclonal. IRDye 800CW donkey antirabbit-IgG (926-32213) and anti-mouse-IgG(H1L) (926-32212) secondary Abs were purchased from LI-COR. Goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary Ab (A0216) was purchased from Beyotime.

Plasmid construction. The plasmids pGEX-4T-1 and pCMV-GFP were used for construction of expression vectors with specific primers (Table S2 in the supplemental material). VP56/VP56-Flag was ligated into pGEX-4T-1 and pCMV-GFP, respectively, to construct expression vectors. Expression plasmids RIG-I-HA, RIG-I-Flag, RIG-I-CARD-HA, RIG-I-helicase-HA, and RIG-I-RD-HA were saved in our lab (41) . For dual-luciferase reporter assays, the valid promoters (RIG-I, IPS-1, STING, TBK1, IRF3, IFN1, IFN3, IFN-g2, and NF-k B1, respectively) were cloned into pGL3 basic luciferase reporter vector (Promega), which had been previously constructed in our lab (41, 55) . HA-Ub, HA-Ub-K63O, and HA-Ub-K48O plasmids were kindly provided by Hong-Bing Shu (Wuhan University, Wuhan, China).

Recombinant expression and purification of VP56. Full-length GCRV-097 VP56 was amplified with corresponding primers (Table S2 ) and cloned into pGEX-4T-1 vector. The plasmid pGEX-4T1-VP56 was transformed into E. coli BL21(DE3) pLysS cells for prokaryotic expression. The fusion protein was induced by isopropyl-b-D-1-thiogalactopyranoside (IPTG) and purified by GST-bind resin (Genscript) chromatography.

Endogenous coimmunoprecipitation, GST pulldown, and LC-MS/MS. Co-IP assays and GST pulldown were performed to explore CIK cell proteins interacting with VP56. For co-IP, CIK cells were infected with GCRV for 24 h, and co-IP was performed with GST Ab, GST-VP56 Ab, or negative serum (NS) using a coimmunoprecipitation kit (Pierce). The eluant was analyzed by SDS-PAGE and subsequent silver staining.

For GST pulldown, CIK cell proteins were extracted with a membrane and cytosol protein extraction kit (Beyotime). Two hundred microliters of GST-VP56 and 200 ml of CIK protein solutions (1 mg/ml, diluted in Tris-buffered saline [TBS]) were incubated at 4°C for 30 min, and then GST-bind resin (20 ml) was added. After 6 h of incubation at 4°C, the resin was washed with TBS thoroughly and eluted with FIG 10 Illustration of VP56 in the regulation of the antiviral signaling pathway in grass carp. Following GCRV-II/ GCRV-III infection, fibrin VP56 and major outer capsid protein VP4 bind together in the cytosol and disperse at the early endosome, lysosome, and ER, etc., where they recruit the viral RNA sensor RIG-I at different regions (VP56 binds to the helicase domain, VP4 binds to CARDs and RD [29] ), form a shield, and obstruct RIG-I sensing of viral RNA. Meanwhile, they synergistically enhance the K48-linked ubiquitination of RIG-I to degrade RIG-I by the proteasomal pathway. Signal transduction from RIG-I to downstream adaptors IPS-1 and STING, TBK1, and IRF3 are then attenuated. Furthermore, VP56 restrains protein expression and phosphorylation of IRF3 and degrades IRF7 (11), thereby inhibiting the subsequent signals of IFNs and NF-k B. Consequently, antiviral effectors are suppressed, and GCRV accomplishes immune evasion and infection. elution buffer (10 mM reduced glutathione and 50 mM Tris-HCl, pH 8.0) and then analyzed using SDS-PAGE and subsequent silver staining.

After co-IP or GST pulldown, LC-MS/MS analysis was respectively performed on a Q Exactive mass spectrometer (Thermo Scientific) by Shanghai Applied Protein Technology Co. Ltd. LC-MS/MS spectra were searched using the MASCOT engine (Matrix Science) against the actinopterygii UniProt sequence database (http://www.uniprot.org/), Grass Carp Genome Database (GCGD) (http://bioinfo.ihb.ac.cn/ gcgd/php/index.php), and a grass carp transcriptome database in the NCBI SRA browser (BioProject accession number SRP049081) (56) .

Western blotting. For Western blotting, protein extracts were separated by 8-12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked in fresh 2% bovine serum albumin (BSA) dissolved in Tris-buffered saline with Tween 20 (TBST) buffer at 4°C overnight and incubated with appropriate indicated primary Abs for 2 h at room temperature. They were then washed three times with TBST buffer and incubated with secondary Ab for 1 h at room temperature. After washing four times with TBST buffer, the nitrocellulose membranes were scanned and imaged by an Odyssey CLx imaging system (LI-COR) or an ImageQuant (GE). The results were performed in triplicate.

Traditional co-IP analysis. For co-IP, CIK cells in 10-cm 2 dishes were cotransfected with the indicated plasmids for 48 h. The cells were lysed in IP lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM Na 3 VO 4 , 0.5 mg/ml leupeptin, and 2.5 mM sodium pyrophosphate) (Beyotime) added with 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice, and the cellular debris was removed by centrifugation at 12,000 Â g for 30 min at 4°C. The supernatant was transferred to a fresh tube and incubated with 1 mg of Ab with gentle shaking overnight at 4°C. Protein A1G Sepharose beads (Beyotime) (30 ml) were added to the mixture and incubated for 2 h at 4°C. After centrifugation at 3,000 Â g for 5 min, the beads were collected and washed four times with lysis buffer. Subsequently, the beads were suspended in 20 ml of 2Â SDS loading buffer and denatured at 95°C for 10 min followed by Western blotting.

Ubiquitination assay. For ubiquitination detection, FHM cells were seeded in dishes and transfected with corresponding plasmids. At 48 h posttransfection, the cells were treated with MG132 (Selleck) for another 6 h. The cells were then harvested for IP with Flag Ab and immunoblotted with HA and Flag Ab, respectively. The experiments were repeated at least three times. The histogram exhibits the relative protein expression levels, which were quantified using ImageJ software.

Far-red mNeptune-based bimolecular fluorescence complementation system. A far-red mNeptunebased bimolecular fluorescence complementation (BiFC) system is used to determine whether two proteins are interactive based on reconstitution of two nonfluorescent fragments of a fluorescent protein (38) . In the present study, the mNeptune-based BiFC system was used to visualize the interaction between VP56 and RIG-I in CIK cells. Briefly, the open reading frame (ORF) sequences of VP56 and RIG-I were amplified and inserted into the pMN155 and pMC156 plasmids, respectively. The final plasmids were named pVP56-MN155 and pMC156-RIG-I, which contain the N-terminal domain of mNeptune (mNeptune amino acids 1 to 155; MN155) behind the C-terminal domain of VP56 and the C-terminal domain of mNeptune (mNeptune amino acids 156 to 244; MC156) in front of the N-terminal domain of RIG-I, respectively. The coding regions were connected by the linker sequence GGGGSGGGGS. Plasmids pVP56-MN155 and pMC156-RIG-I were then transfected into CIK cells alone or together as described above. mNeptune was observed by confocal microscopy. Red mNeptune BiFC signals were measured with excitation at 640/20 nm and emission at 685/40 nm.

Dual-luciferase reporter assay. CIK cells were seeded in 24-well plates for 24 h. Cotransfection was performed with corresponding expression plasmid, target promoter luciferase plasmid, and internal control reporter vector (pRL-TK). At 24 h posttransfection, cells were infected with GCRV or uninfected for 24 h. The cells were washed with phosphate-buffered saline (PBS) and lysed with passive lysis buffer (Promega) for 30 min. Luciferase activities were detected by a dual-luciferase reporter assay system (Promega). The luciferase reading was normalized against those in the pRL-TK levels, and the relative light unit intensity was presented as the ratio of luciferase of firefly to renilla.

Transcriptome analyses and qRT-PCR verification. Transcriptome data of empty vector stably transfected or VP56 stably transfected CIK cells were derived from previous studies performed by our lab and deposited in transcriptome databases (NCBI SRA number SRP212372, sample SAMN12168356/SAMN12168358) (29) . Transcriptome data were verified for the effect of VP56 on the RLR signaling pathway and related IFN responses at the mRNA level. Gene expression levels according to the transcriptome data were validated by qRT-PCR.

Crystal violet staining. For crystal violet staining, CIK cells were seeded into 24-well plates overnight and transiently transfected with empty vector or VP56. At 24 h posttransfection, cells were infected with GCRV or uninfected. At 24 h postinfection, cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.05% (wt/vol) crystal violet (Sigma, USA) for 30 min then washed with water and drained. Finally, the plates were photographed under a light box (Bio-Rad).

siRNA-mediated knockdown of VP56. CIK cells stably expressing VP56 were used for VP56 knockdown assays. Transient knockdown of VP56 was achieved by transfection of siRNA targeting VP56 mRNA. Three siRNA sequences (si-VP56-1 [sense 59 to 39], CUCCACAACUUUAGAUGAATT, si-VP56-2 [sense 59 to 39], CCUAUAGCCGUCGCUAAAUTT, and si-VP56-3 [sense 59 to 39], GGAGGAAGCAUUUGUAGGUTT) targeting different regions of VP56 were synthesized by GenePharma (Jiangsu, China). CIK cells were transfected with siRNA using GP-siRNA-Mate Plus (GenePharma, China) for 24 h. Silencing efficiencies of the candidate siRNAs were evaluated by qRT-PCR, and results were compared with those in the negative-control siRNA (si-NC) provided by the supplier. A preliminary experiment indicated that si-VP56-1 possessed the best silencing efficiency at a final concentration of 100 nM at the mRNA level. For Western blotting, CIK cells stably expressing

VP56 were plated in 6-well plates and transfected with si-VP56-1 using GP-siRNA-Mate Plus (GenePharma, China) for 24 h and infected with GCRV for another 24 h. The cells were lysed, and proteins were extracted for Western blotting

3.1 ml of nucleasefree water, 4 ml of diluted cDNA (200 ng), and 0.2 ml of each gene-specific primer (10 mM) (Table S2). Statistical analysis. The data were analyzed as previously described (58). Briefly, data were analyzed using an unpaired, two-tailed Student's t test

Cyprinid viral diseases and vaccine development

Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses

Complete genome sequence of a reovirus isolated from grass carp, indicating different genotypes of GCRV in China

Identification of a novel membraneassociated protein from the S7 segment of grass carp reovirus

Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae)

Complete genomic sequence of a reovirus isolated from grass carp in China

Backbone model of an aquareovirus virion by cryo-electron microscopy and bioinformatics

Prediction of GCRV virus-host protein interactome based on structural motif-domain interactions

Bioinformatics of recent aqua-and orthoreovirus isolates from fish: evolutionary gain or loss of FAST and fiber proteins and taxonomic implications

Grass carp reovirus-GD108 fiber protein is involved in cell attachment

Grass carp reovirus VP56 represses interferon production by degrading phosphorylated IRF7

Cytosolic sensing of viruses

Insights into the antiviral immunity against grass carp (Ctenopharyngodon idella) reovirus (GCRV) in grass carp

Sensing disease and danger: a survey of vertebrate PRRs and their origins

IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction

Type I interferon gene induction by the interferon regulatory factor family of transcription factors

Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes

Innate immunity of finfish: primordial conservation and function of viral RNA sensors in teleosts

Advances in aquatic animal RIG-I-like receptors. Fish and Shellfish Immunol Rep 2:100012

Expression and functional characterization of the RIG-I-like receptors MDA5 and LGP2 in rainbow trout (Oncorhynchus mykiss)

Origin and evolution of the RIG-I like RNA helicase gene family

Progresses on three pattern recognition receptor families (TLRs, RLRs and NLRs) in teleost

Molecular characterization of IRF3 and IRF7 in rainbow trout, Oncorhynchus mykiss: functional analysis and transcriptional modulation

Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/ MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway

Characterization of fish IRF3 as an IFN-inducible protein reveals evolving regulation of IFN response in vertebrates

Endogenous viruses: insights into viral evolution and impact on host biology

Pacing a small cage: mutation and RNA viruses

A viral deamidase targets the helicase domain of RIG-I to block RNA-induced activation

Grass carp reovirus major outer capsid protein VP4 interacts with RNA sensor RIG-I to suppress interferon response

Junctional adhesion molecule A serves as a receptor for prototype and field-isolate strains of mammalian reovirus

Junctional adhesion molecule-A is required for hematogenous dissemination of reovirus

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp reovirus NS26 interacts with cellular lipopolysaccharide-induced tumor necrosis factor-alpha factor

Sequestration of RNA by grass carp Ctenopharyngodon idella TIA1 is associated with its positive role in facilitating grass carp reovirus infection

Laminin receptor is an interacting partner for viral outer capsid protein VP5 in grass carp reovirus infection

Grass carp Ctenopharyngodon idella Fibulin-4 as a potential interacting partner for grass carp reovirus outer capsid proteins

Orthoreovirus outer-fiber proteins are substrates for SUMO-conjugating enzyme Ubc9

In vivo imaging of protein-protein and RNAprotein interactions using novel far-red fluorescence complementation systems

Structural insights into RNA recognition and activation of RIG-I-like receptors

RIG-I-like receptor regulation in virus infection and immunity

Grass carp laboratory of genetics and physiology 2 serves as a negative regulator in retinoic acid-inducible gene I-and melanoma differentiation-associated gene 5-mediated antiviral signaling in resting state and early stage of grass carp reovirus infection

Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity

Ubiquitin-mediated modulation of the cytoplasmic viral RNA sensor RIG-I

Structural and functional views of the intracellular viral RNA sensor RIG-I

Broad-spectrum robust direct bactericidal activity of fish IFNw 1 reveals an antimicrobial peptide-like function for type I IFNs in vertebrates

Ten strategies of interferon evasion by viruses

Regulation of RIG-I-like receptor-mediated signaling: interaction between host and viral factors

Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity

Dengue virus targets the adaptor protein MITA to subvert host innate immunity

Innate immunity evasion by dengue virus

Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling

Evasion of antiviral immunity through sequestering of TBK1/IKK« /IRF3 into viral inclusion bodies

PLP2 of mouse hepatitis virus A59 (MHV-A59) targets TBK1 to negatively regulate cellular type I interferon signaling pathway

Hepatitis C virus NS2 protease inhibits host cell antiviral response by inhibiting IKK« and TBK1 functions

MDA5 induces a stronger interferon response than RIG-I to GCRV infection through a mechanism involving the phosphorylation and dimerization of IRF3 and IRF7 in CIK cells

Transcriptome analysis provides insights into the regulatory function of alternative splicing in antiviral immunity in grass carp

A plasmid containing CpG ODN as vaccine adjuvant against grass carp reovirus in grass carp Ctenopharyngodon idella

A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella

The authors highly appreciate Hong-Bing Shu (Wuhan University, Wuhan, China) for providing plasmids ( 

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1.2 MB.