Mitochondrial antiviral signaling protein plays a major role in induction of the fish innate immune response against RNA and DNA viruses

Abstract

Viral infection triggers host innate immune responses through cellular sensor molecules which activate multiple signaling cascades that induce the production of interferons (IFN) and other cytokines. The recent identification of mammalian cytoplasmic viral RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and their mitochondrial adaptor, the mitochondrial antiviral signaling protein (MAVS), also called IPS-1, VISA, and Cardif, highlights the significance of these molecules in the induction of IFN. Teleost fish also possess a strong IFN system, but nothing is known concerning the RLRs and their downstream adaptor. In this study, we cloned MAVS cDNAs from several fish species (including salmon and zebrafish) and showed that they were orthologs of mammalian MAVS. We demonstrated that overexpression of these mitochondrial proteins in fish cells led to a constitutive induction of IFN and IFN-stimulated genes (ISGs). MAVS-overexpressing cells were almost fully protected against RNA virus infection, with a strong inhibition of both DNA and RNA virus replication (1,000- and 10,000-fold decreases, respectively). Analyses of MAVS deletion mutants showed that both the N-terminal CARD-like and C-terminal transmembrane domains, but not the central proline-rich region, were indispensable for MAVS signaling function. In addition, we cloned the cDNAs encoding a RIG-I-like molecule from salmonid and cyprinid cell lines. Like the case with MAVS, overexpression of RIG-I CARDs in fish cells led to a strong induction of both IFN and ISGs, conferring on fish cells full protection against RNA virus infection. This report provides the first demonstration that teleost fish possess a functional RLR pathway in which MAVS may play a central role in the induction of the innate immune response.

FIG. 1.

MAVS sequences through vertebrate evolution. (A) Multiple alignment of MAVS sequences from fish and other vertebrates. The CARD and the intramembrane (IM) domain are indicated above the alignment. Relevant positions conserved in the CARD are boxed in gray. The residues of the TM region, predicted by the TMpred server, are shown in bold. The region missing in a salmon transcript, probably due to alternate splicing, is underlined. This salmon MAVS splicing variant is called mini-MAVS. The little skate (Leucoraja erinacea) sequence is from GenBank accession no. DT726507, and the lamprey (Petromyzon marinus) sequence is from GenBank accession no. FD727562. (B) Maximum likelihood phylogenetic tree of vertebrate MAVS. Neighbor-joining analysis led to a similar tree (not shown). Sequences from the following organisms (GenBank accession number, unless indicated otherwise) were included: Atlantic salmon (FN178458; this study), Salmo salar; zebrafish (FN178460; this study), Danio rerio; EPC cell line (FN178455; this study); medaka fish (AM299285 and Ensembl no. Chr22_25852569), Oryzia latipes; fugu (Ensembl release 52 no. scaffold338_161560), Fugu rubripes; pufferfish (Ensembl release 52 no. Chr10_12331965), Tetraodon nigroviridis; stickleback (Ensembl no. LG II_11620643), Gasterosteus aculeatus; chicken (NP_001012911), Gallus gallus; mouse (NP_659137), Mus musculus; and human (NP_065797), Homo sapiens. (C) Conserved synteny around the MAVS gene in zebrafish, medakas, mice, and humans. The locations of the different markers and the chromosomes involved are indicated for the different species (data from the assemblies are available at the Ensembl website [http://www.ensembl.org/]).

FIG. 2.

Localization of salmon MAVS to mitochondria. peGFP-MAVS or peGFP, as a control, was transfected into EPC cells. The mitochondria and the nuclei were stained in vivo with MitoTracker (red) and Hoechst (blue), respectively, and the cells were imaged by microscopy. The yellow staining in the overlay image indicates colocalization of MAVS and MitoTracker.

FIG. 3.

Fish MAVS are strong antiviral proteins. CHSE 214 (A) and EPC (B) cells were transfected with 2 μg of pcDNA-MAVS, peGFP-MAVS, or an empty vector (pcDNA) as a control. At 24 h and 6 days posttransfection, EPC and CHSE 214 cells, respectively, were infected with VHSV at an MOI of 5 for 5 days at 15°C. Cell monolayers were then stained with crystal violet. The culture supernatants from cells infected with VHSV were collected at 0 and 4 days postinfection (dpi) for CHSE 214 cells (C) and at 48 h postinfection (hpi) for EPC cells (D), and the viral titer was determined by plaque assay on EPC cells. Each time point was represented by two independent experiments, and each virus titration was done in duplicate. Means are shown. The standard errors were calculated, but the bars are not shown because the errors were very small. (E) CHSE 214 cells were transfected with 2 μg of pcDNA-MAVS or an empty vector (pcDNA) as a control. At 1, 3, or 6 days posttransfection (dpt), CHSE 214 cells were infected with VHSV at an MOI of 5. The culture supernatants were collected at 4 days postinfection, and the viral titer was determined by plaque assay on EPC cells. (F) EPC cells were transfected with 2 μg of pcDNA vector encoding MAVS from salmon (Sal), zebrafish (Zf), or EPC cells or an empty vector (pcDNA) as a control. At 48 h posttransfection, EPC cells were infected with VHSV at an MOI of 5 for 6 days at 15°C. Cell monolayers were then stained with crystal violet, and the viral titer was determined for each culture supernatant by plaque assay.

FIG. 4.

Overexpression of MAVS induces antiviral immunity against several viruses. EPC cells were transfected with 2 μg of pcDNA-MAVS or an empty vector (pcDNA) as a control. At 24 h posttransfection, EPC cells were infected with different RNA viruses of the Rhabdoviridae family, i.e., VHSV, IHNV, and SVCV, at an MOI of 5 (A) or with a DNA virus of the Iridoviridae family, i.e., EHNV, at an MOI of 2 (B). The culture supernatants were collected at 48 h or 24 h postinfection, respectively, and the viral titer was determined by plaque assay on EPC cells. For panel B, cell monolayers were then stained with crystal violet. Each time point was represented by two independent experiments, and each virus titration was done in duplicate. Means are shown.

FIG. 5.

The CARD-like domain and the mitochondrial membrane targeting sequence are essential for MAVS signaling. (A) Diagram illustrating the MAVS splicing variant and mutants. Mini, salmon MAVS splicing variant lacking residues 126 to 553; ΔCARD, salmon MAVS mutant lacking the CARD-like domain (residues 2 to 93); ΔTM, mutant lacking the mitochondrial membrane targeting sequence (residues 568 to 606). On the right is the size of each protein, in amino acids. (B) EPC cells were transfected with 2 μg of pcDNA vector encoding MAVS, mini-MAVS, and its mutants (ΔCARD and ΔTM). At 24 h posttransfection, the cells were infected with VHSV at an MOI of 5. The culture supernatants were collected at 48 h postinfection, and the viral titer was determined by plaque assay. The cell monolayers were stained with crystal violet at 5 days postinfection.

FIG. 6.

CARD-like domains of fish RIG-I and MAVS. (A) Multiple alignment of RIG-I CARDs from fish and other vertebrates. The conserved residues are boxed in gray. The CARDs and the beginning of the helicase domain are mapped. Sequences for the following (GenBank accession numbers) were used: salmon RIG-I (FN178459; this study), from Salmo salar; EPC RIG-I (FN178456; this study); mouse RIG-I (NP_766277), from Mus musculus; and human RIG-I (NP_055129), from Homo sapiens. (B) Neighbor-joining phylogenetic tree of vertebrate CARDs from RIG-I, MDA5, and MAVS. The tree was calculated by ClustalW, based on a multiple alignment of RIG-I, MDA5, and MAVS CARD-like domains from fish and other vertebrates (see Fig. S1 in the supplemental material), and a 1,000-bootstrap analysis was performed. High bootstrap values (>800) are indicated. The following sequences (GenBank accession number, unless indicated otherwise) were used: salmon RIG-I (FN178459; this study) and MAVS (FN178458; this study), from Salmo salar; zebrafish RIG-I (Ensembl protein no. ENSDARP00000058175; gene located at Chr23 positions 46,283,361 to 46,298,826), MDA5 (NC_007120), and MAVS (FN178460; this study), from Danio rerio; EPC RIG-I (FN178456; this study) and MAVS (FN178455; this study); mouse RIG-I (NP_766277), MDA5 (AAM21359), and MAVS (NP_659137), from Mus musculus; and human RIG-I (NP_055129), MDA5 (AAG34368), and MAVS (NP_065797), from Homo sapiens.

FIG. 7.

Overexpression of MAVS or the N-terminal region of RIG-I induces powerful antiviral immunity. (A) EPC cells were transfected with 2 μg of pcDNA vector encoding MAVS, RIG-I Nter (first 275 aa), or the IFN from EPC cells (FN178457; this study) or an empty vector (pcDNA) as a control. At 24 h posttransfection, EPC cells were infected with VHSV at an MOI of 5 for 6 days at 15°C. Cell monolayers were then stained with crystal violet. The UV-visible light microscope picture on the right shows the level of RIG-I Nter-GFP fusion protein expression achieved in EPC cells at 24 h posttransfection. (B) The viral titer for each culture supernatant was determined by plaque assay at 48 h postinfection. Each time point is represented by two independent experiments, and each virus titration was done in duplicate. Means are shown. (C) The culture supernatants of mock-infected wells were harvested at 4 days posttransfection, and the protective effects of the IFNs secreted after overexpression of MAVS, CARDs of RIG-I (RIG-I Nter), and IFN itself were measured by monitoring the inhibition of CPE caused by VHSV.

FIG. 8.

Overexpression of MAVS and RIG-I induces IFN and ISG expression. EPC cells were transfected with 2 μg of pcDNA vector encoding salmon MAVS or RIG-I Nter (first 275 aa) or an empty vector (pcDNA) as a control. At 24 h posttransfection, EPC cells were infected or not with VHSV at an MOI of 5 and incubated at 15°C for 12 and 24 h before total RNA extraction and RT. Quantitative real-time PCR was conducted using primers targeting RIG-I (A), MAVS (B), IFN (C), VIG-1 (D), ISG15-1 (E), and ISG15-2 (F) (see Table S1 in the supplemental material). The β-actin gene was used as an internal control to normalize the cDNA template and for real-time PCR calculations. The standard deviations for triplicate experiments are shown.