Zebrafish ISG15 exerts a strong antiviral activity against RNA and DNA viruses and regulates the interferon response

Abstract

ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.

Fig 1

Characterization of zf-ISG15 expression in vivo and in vitro. (A) zf-ISG15 contains two ubiquitin-like domains (UBL) and a C-terminal peptide with a diglycine motif directly involved in ISG15 conjugation to the target proteins. The level of protein sequence identity between ISG15 from different species is indicated on the right. (B) zf-isg15 is induced upon viral infection. Adult zebrafish were infected by IHNV 25-70 or SVCV, and zf-isg15 gene expression in the gut of infected or mock-infected animals was measured by qPCR. Transcript copy numbers were normalized to the level of β-actin expression (measured ratio of mRNA of interest/β-actin mRNA). The means and SDs of three experiments are shown. Significant differences determined using Welch's unpaired t test are indicated (*, P = 0.05). (C) HA-zf-ISG15 (the free and conjugated isoforms are indicated on the right) was detected in cell lysates of pHA-zf-ISG15-transfected EPC cells (lane 2) after anti-HA immunoblotting, while no signal was detected from mock-transfected cells (lane 1). In contrast, HA-zf-ISG15_LRAA mutant expression was restricted to free isoforms (lane 3). α-TUB, α-tubulin.

Fig 2

Inhibition of different RNA and DNA viruses by zf-ISG15 overexpression. (A) Kinetics of viral growth measured by viral titration from 0 to 72 hpi in the supernatant of EPC cells transfected with pHA-zf-ISG15_LRGG (dark blue), pHA-zf-ISG15_LRAA (red), GFP (green), or an empty vector (violet) or mock transfected (blue) and infected with the indicated RNA and DNA viruses at an MOI of 1. The means and SDs of three independent experiments are shown; the error bars are not always visible when the differences are very small. The x axis shows the times (hours) posttransfection, and the y axis shows the virus titers (PFU). (B) Western blot analysis of viral protein expression under zf-ISG15 overexpression. EPC cells were transfected with zf-ISG15 or empty vector before infection with IHNV 25-70 at an MOI of 1 for 24 h. Viral protein expression was evaluated by immunoblotting using anti-IHNV polyclonal antibody (α-IHNV), while ISG15 expression was detected by using anti-HA antibody (α-HA). α-Tubulin was used as a loading control. Lanes: 1, empty vector plus IHNV 25-70; 2, empty vector; 3, HA-zf-ISG15 plus IHNV 25-70; 4, HA-zf-ISG15. (C) Cytopathic effects of different viruses at 72 hpi on a lawn of EPC cells at an MOI 1, assessed by staining of viable cells by crystal violet. Cells had previously been transfected with pHA-zf-ISG15 (ISG15_LRGG), pHA-zf-ISG15_LRAA (ISG15_LRAA), GFP, or an empty vector (vector) or were mock transfected (mock). NI, noninfected cells presented as a control.

Fig 3

ISG15 conjugation targets viral proteins. (A) ISGylated proteins were immunoprecipitated with agarose-immobilized anti-HA antibody before anti-HA immunodetection from EPC cells overexpressing HA-zf-ISG15 infected (lane 1) or not (lane 2) with IHNV 25-70. (B) ISG15 binding to IHNV proteins was evaluated in EPC cell extracts at 72 hpt with expression plasmid pHA-zf-ISG15 alone or combined with IHNV NV-GFP or P-GFP. The expression level of the tagged proteins was determined from total cell lysates (crude extracts) by Western blotting using anti-HA antibody (α-HA), anti-GFP antibody (α-GFP), and α-tubulin (α-TUB) as a gel loading control. Native P and NV proteins are indicated by asterisks on the right. ISGylation of the viral proteins was then visualized by anti-HA immunodetection of proteins coimmunoprecipitated with GFP antibody (GFP-IP) after two different exposure times. Lanes: 1, pHA-zf-ISG15; 2, P-GFP; 3, NV-GFP; 4, pHA-zf-ISG15 plus P-GFP; 5, pHA-zf-ISG15 plus NV-GFP (lane 5). *, molecular masses of native viral proteins. ISG15 conjugates are indicated on the right.

Fig 4

The LRAA motif is required for ISGylation of cellular and viral proteins. (A) Coimmunoprecipitation of HA-zf-ISG15 and zf-TRIM25-V5 with agarose-immobilized anti-HA antibody (HA-IP), prior to Western blot detection of immunoprecipitated proteins using anti-HA (α-HA) antibody. The level of expression of tagged proteins in cell lysate at 72 hpt (crude extract) was evaluated by Western blotting using anti-HA or anti-V5 antibodies and normalized with α-tubulin. *, position of native HA-ISG15 or TRIM25-V5 protein. ISGylated conjugates are indicated on the right. Lanes: 1, HA-zf-ISG15; 2, HA-zf-ISG15_LRAA; 3, zf-TRIM25-V5; 4, HA-zf-ISG15 plus zf-TRIM25-V5; 5, HA-zf-ISG15_LRAA plus zf-TRIM25-V5. ISG15_LRAA does not interact with the IHNV NV (B) or P (C) protein. ISGylated proteins were immunodetected with anti-HA antibody after immunoprecipitation of EPC cell extracts with anti-GFP antibody (GFP-IP). Evaluation of the level of expression of tagged proteins was performed by Western blotting on total cell lysate (crude extracts) using anti-GFP (α-GFP) and anti-HA (α-HA) antibodies, and normalization was performed with α-tubulin detection. (B) pHA-zf-ISG15 (lane 1), pHA-zf-ISG15_LRAA (lane 2), NV-GFP (lane 3), GFP (lane 4), pHA-zf-ISG15 plus GFP (lane 5), pHA-zf-ISG15 plus NV-GFP (lane 6), pHA-zf-ISG15_LRAA plus NV-GFP (lane 7); (C) pHA-zf-ISG15 (lane 8), pHA-zf-ISG15_LRAA (lane 9), P-GFP (lane 10), GFP (lane 11), pHA-zf-ISG15 plus GFP (lane 12), pHA-zf-ISG15 plus P-GFP (lane 13), pHA-zf-ISG15_LRAA plus P-GFP (lane 14). *, molecular masses of native GFP or viral proteins.

Fig 5

Anti-P immunoprecipitation supports ISGylation of the INHV P protein. ISGylated proteins were immunodetected with anti-HA antibody after immunoprecipitation of EPC cell extracts with anti-IHNV P antibody (P_IHNV-IP). Evaluation of the level of P protein expression was performed by Western blotting on total cell lysate (crude extracts) using anti-IHNV (α-IHNV) and anti-HA (α-HA) antibodies, and normalization was performed with α-tubulin detection. Lanes: 1, pHA-zf-ISG15; 2, pHA-zf-ISG15 plus INHV P; 3, INHV P. *, molecular mass of native P; plain arrow, mono-ISGylated conjugates of the P protein; dotted arrow, Ig heavy chain.

Fig 6

ISG15 modulates expression of the IFN-ϕ gene, vig-1/viperin, and rig-I. Expression of the IFN-ϕ gene, vig-1/viperin, rig-I, and Ef1-α, in EPC cells overexpressing HA-zf-ISG15_LRGG or HA-zf-ISG15_LRAA was measured by qPCR at 72 hpt. IFN-ϕ gene, vig-1/viperin, rig-I, and Ef1-α transcript copy numbers were normalized to the level of β-actin expression (measured ratio of mRNA of interest/β-actin mRNA). Means and SDs of the fold induction or repression are shown for triplicates of a representative experiment. Significant differences were determined using Welch's unpaired t test performed on ΔΔC_T values (where C_T is the threshold cycle) (**, P = 0.01).