Dataset Information

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response.

ABSTRACT: Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/MS, and then verified by co-IP and an advanced far-red fluorescence complementation system. VP4 was proved to bind to the CARD and RD domains of RIG-I and promoted K48-linked ubiquitination of RIG-I to degrade RIG-I. VP4 reduced mRNA and promoter activities of key genes of RLR pathway and sequential IFN production. As a consequence, antiviral effectors were suppressed and GCRV replication increased, resulting in intensified cytopathic effect. Furthermore, results of transcriptome sequencing of VP4 stably expressed CIK (C. idella kidney) cells indicated that VP4 activated the MyD88-dependent TLR pathway. Knockdown of VP4 obtained opposite effects. These results collectively revealed that VP4 interacts with RIG-I to restrain interferon response and assist GCRV invasion. This study lays the foundation for anti-dsRNA virus molecular function research in teleost and provides a novel insight into the strategy of immune evasion for aquatic virus.

SUBMITTER: Su H

PROVIDER: S-EPMC7226501 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response.

Su Hang H Fan Chengjian C Liao Zhiwei Z Yang Chunrong C Clarke Jihong Liu JL Zhang Yongan Y Su Jianguo J

Biomolecules 20200406 4

Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/M ...[more]

PMID: 32268551

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Project description: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.

Project description:BackgroundMyxovirus resistance (Mx) proteins are crucial effectors of the innate antiviral response against a wide range of viruses, mediated by the type I interferon (IFN-I) signaling pathway. However, the antiviral activity of Mx proteins is diverse and complicated in different species.Methodology/principal findingsIn the current study, two novel Mx genes (CiMx1 and CiMx3) were identified in grass carp (Ctenopharyngodon idella). CiMx1 and CiMx3 proteins exhibit high sequence identity (92.1%), and low identity with CiMx2 (49.2% and 49.5%, respectively) from the GenBank database. The predicted three-dimensional (3D) structures are distinct among the three isoforms. mRNA instability motifs also display significant differences in the three genes. The spatial and temporal expression profiles of three C. idella Mx genes and the IFN-I gene were investigated by real-time fluorescence quantitative RT-PCR (qRT-PCR) following infection with grass carp reovirus (GCRV) in vivo and in vitro. The results demonstrated that all the four genes were implicated in the anti-GCRV immune response, that mRNA expression of Mx genes might be independent of IFN-I, and that CIK cells are suitable for antiviral studies. By comparing expression patterns following GCRV challenge or poly(I:C) treatment, it was observed that GCRV blocks mRNA expression of the four genes. To determine the functions of Mx genes, three CiMx cDNAs were cloned into expression vectors and utilized for transfection of CIK cells. The protection conferred by each recombinant CiMx protein against GCRV infection was evaluated. Antiviral activity against GCRV was demonstrated by reduced cytopathic effect, lower virus titer and lower levels of expressed viral transcripts. The transcription of IFN-I gene was also monitored.Conclusions/significanceThe results indicate all three Mx genes can suppress replication of grass carp reovirus and over-expression of Mx genes mediate feedback inhibition of the IFN-I gene.

Project description:BackgroundThe grass carp hemorrhagic disease caused by the grass carp reovirus (GCRV) is a major disease that hampers the development of grass carp aquaculture. The mechanism underlying GCRV pathogenesis and hemorrhagic symptoms is still unknown. MicroRNAs (miRNAs) are key regulators involved in various biological processes. The aim of this study was to identify conserved and novel miRNAs in grass carp in response to GCRV infection, as well as attempt to reveal the mechanism underlying GCRV pathogenesis and hemorrhagic symptoms.ResultsGrass carp were infected with GCRV, and spleen samples were collected at 0 (control), 1, 3, 5, 7, and 9 days post-infection (dpi). These samples were used to construct and sequence small RNA libraries. A total of 1208 miRNAs were identified, of which 278 were known miRNAs and 930 were novel miRNAs. Thirty-six miRNAs were identified to exhibit differential expression when compared with the control, and 536 target genes were predicted for the 36 miRNAs. GO and KEGG enrichment analyses of these target genes showed that many of the significantly enriched terms were associated with immune response, blood coagulation, hemostasis, and complement and coagulation cascades, especially the GO term "blood coagulation" and pathway "complement and coagulation cascades." Ten representative target genes involved in "complement and coagulation cascades" were selected for qPCR analysis, and the results showed that the expression patterns of these target genes were significantly upregulated at 7 dpi, suggesting that the pathway "complement and coagulation cascades" was strongly activated.ConclusionConserved and novel miRNAs in response to GCRV infection were identified in grass carp, of which 278 were known miRNAs and 930 were novel miRNAs. Many of the target genes involved in immune response, blood coagulation, hemostasis, and complement and coagulation cascades. Strong activation of the pathway "complement and coagulation cascades" may have led to endothelial-cell and blood-cell damage and hemorrhagic symptoms. The present study provides a new insight into understanding the mechanism underlying GCRV pathogenesis and hemorrhagic symptoms.

Dataset Information

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response.

Publications

Grass Carp Reovirus Major Outer Capsid Protein VP4 Interacts with RNA Sensor RIG-I to Suppress Interferon Response.

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