Zebrafish maoc1 Attenuates Spring Viremia of Carp Virus Propagation by Promoting Autophagy-Lysosome-Dependent Degradation of Viral Phosphoprotein

ABSTRACT

Spring viremia of carp virus (SVCV) is the causative agent of spring viremia of carp (SVC), an important infectious disease that causes high mortality in aquaculture cyprinids. How the host defends against SVCV infection and the underlying mechanisms are still elusive. In this study, we identify that a novel gene named maoc1 is induced by SVCV infection. maoc1-deficient zebrafish are more susceptible to SVCV infection, with higher virus replication and antiviral gene induction. Further assays indicate that maoc1 interacts with the P protein of SVCV to trigger P protein degradation through the autophagy-lysosomal pathway, leading to the restriction of SVCV propagation. These findings reveal a unique zebrafish defense machinery in response to SVCV infection.

IMPORTANCE SVCV P protein plays an essential role in the virus replication and viral immune evasion process. Here, we identify maoc1 as a novel SVCV-inducible gene and demonstrate its antiviral capacity through attenuating SVCV replication, by directly binding to P protein and mediating its degradation via the autophagy-lysosomal pathway. Therefore, this study not only reveals an essential role of maoc1 in fighting against SVCV infection but also demonstrates an unusual host defense mechanism in response to invading viruses.

INTRODUCTION

Epidemics of fish viral diseases cause tremendous losses to aquaculture worldwide every year (1). Spring viremia of carp (SVC) is one of the important fish viral diseases affecting cyprinids and causing significant morbidity and mortality of aquaculture fish in Europe, America, and Asian countries (2, 3). The causative virus of this disease is spring viremia of carp virus (SVCV), a member of the genus Vesiculovirus of the family Rhabdoviridae. SVCV is a negative-strand RNA virus (NSV), whose genome contains five genes in the order 3′-N-P-M-G-L-5′, encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L), respectively (1, 4). Infected fish exhibit complex and mixed symptoms, such as hemorrhage of the skin, gills, eyes, and internal organs and degeneration of the gill lamellae.

In order to understand the pathogenesis of SVCV infection and fish defense against SVCV for the prevention and control of this disease, multiple studies have been conducted from different aspects. On the one hand, it is demonstrated that the N protein of SVCV mediates degradation of the adaptor protein MAVS via the ubiquitin-proteasome pathway to block the host interferon (IFN) production, leading to the facilitation of SVCV propagation (5). The P protein of SVCV inhibits TBK1 activity, resulting in decreasing interferon regulatory factor 3 (IRF3) phosphorylation and subsequently suppressing IRF3 transcriptional activity (6). On the other hand, multiple pieces of evidence suggest that fish factors can indirectly influence SVCV propagation by negatively or positively modulating host antiviral innate immune responses (4–11). However, whether fish factors can act on the proteins of SVCV to directly affect SVCV propagation is still largely unknown.

In fact, in mammals, it has been well defined that the host factors can directly act on viral factors to limit or facilitate virus replication. DExD-box helicase 21 (DDX21) RNA helicase inhibits polymerase assembly of influenza A by binding to PB1, leading to decreasing viral RNA and protein synthesis and, subsequently, restricting influenza A replication (12). SCOTIN, an IFN-β-induced host protein, recruits the nonstructural protein 5A (NS5A) protein of hepatitis C virus (HCV) onto autophagosome for degradation, resulting in the inhibition of HCV replication (13). Human tripartite motif containing 26 (TRIM26) interacts with the nonstructural protein 5B (NS5B) protein of HCV to mediate its K27-linked ubiquitination and facilitate subsequent NS5B-NS5A interaction, thereby promoting HCV replication (14). Obviously, to understand how host factors directly act on viral factors to influence virus propagation will benefit the development of new treatments and prevention of virus infection.

Due to the convenience of genetic manipulation, zebrafish has become a widely used model for different research purposes (15). Interestingly, zebrafish also belongs to the cyprinids and can be infected by SVCV. Thus, it has been utilized as a model to study SVCV infection (7–11). In this study, in order to understand how fish fight against invading SVCV, we took advantage of the zebrafish model and identified that zebrafish maoc1 attenuates SVCV propagation by promoting autophagy-lysosome-dependent degradation of viral phosphoprotein, revealing a unique defense mechanism of fish against SVCV infection.

RESULTS

Zebrafish maoc1 is induced by SVCV infection.

As reported previously, SVCV can infect zebrafish to cause typical symptoms of SVC disease and induce antiviral gene expression (4–11). In this study, initially, we confirmed that SVCV propagated in zebrafish larvae as indicated by the expression of G, P, and N protein gene mRNA (see Fig. S1A to C in the supplemental material). In addition, the typical antiviral genes, including ifn1, lta, and mxc, and inflammatory cytokine genes, including il1β, il6, and tnfα, were induced by SVCV infection (Fig. S1D to I). Subsequently, to determine the defense mechanisms of fish in response to SVCV infection, we screened SVCV-induced genes in zebrafish larvae. Among the SVCV-induced genes, a novel gene, si:dkey-208 k22.6 (ensemble accession number ENSDARG00000061800), got our attention and was induced dramatically by SVCV infection in a dose-dependent manner, similar to the levels of the typical antiviral gene pkz and cytokine gene il6 (Fig. 1A and B). Sequencing alignment indicates that this gene contains a conserved MARVEL domain and an occludin domain (Fig. S2), and we thus named it maoc1 (MARVEL domain and occludin domain-containing protein 1). Zebrafish maco1 is highly expressed in liver and kidney (Fig. 1C). Therefore, we identify an SVCV-induced novel gene, maco1, in zebrafish.

FIG 1.

Zebrafish maoc1 is upregulated upon SVCV infection; generation of maoc1-null zebrafish via CRISPR/Cas9 techniques. (A) Zebrafish maoc1 as well as pkz, ifn1, and il6 mRNA was induced by SVCV (virus strain OMG067) infection (~2.5 × 10⁷ TCID₅₀/mL) in ZFL cells. ZFL cells were seeded in 60-mm plates overnight and infected with SVCV; total RNA was extracted for examining the expression of target genes by quantitative real-time PCR (qRT-PCR). (B) The expression of zebrafish maoc1 mRNA in zebrafish larvae (3 days postfertilization [3 dpf]) was induced by SVCV infection (0, 0.5, 1, 2.5 × 10⁷ TCID₅₀/mL) in a dose-dependent manner. (C) The mRNA levels of maoc1 in heart, brain, liver, spleen, kidney, ovary, spermary, muscle, and eye of zebrafish (3 mpf). (D) Verification of the efficiency of CRISPR/Cas9-mediated zebrafish maoc1 disruption by heteroduplex mobility assay (HMA) and schematic of the targeting site in maoc1 and the resulting nucleotide sequence in the mutant (MT, maoc1^{hbsynm1/ihbsynm1}). The maoc1^−/− zebrafish were obtained by mating maoc1^+/– (♀) and maoc1^+/– (♂). (E) The predicted protein product of maoc1 in the mutant and wild-type (WT) sibling. (F) The relative mRNA levels of maoc1 in the WT and homozygous mutant. (G) The maoc1 protein in liver of WT and mutant zebrafish (3 mpf). All data are presented as mean values based on three repeated experiments, and error bars indicate the ±SEM. *, P < 0.05, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

maoc1-null zebrafish are more susceptible to SVCV infection.

To determine the role of maoc1 in vivo, we knocked out maoc1 in zebrafish using the CRISPR/Cas9 and obtained one mutant line with a 5-nucleotide deletion in exon 2 of maoc1 (maoc1^{ihbsynm1/ihbsynm1}) (Fig. 1D and E). A quantitative real-time PCR (qRT-PCR) assay and immunoblot analysis confirmed that maoc1 was disrupted efficiently. By crossing maoc1^+/– (♀) and maoc1^+/– (♂), the offspring with maoc1^+/+, maoc1^+/–, and maoc1^−/− genetic backgrounds were born at a Mendelian ratio (1:2:1), and no obvious defects in growth rate and reproduction capability were detected in maoc1^−/− zebrafish under normal conditions. We challenged maoc1^−/− larvae (4 days postfertilization [dpf]) and maoc1^+/+ larvae (4 dpf) (having a wild-type allele of maoc1) (WT) with high-titer SVCV and photographed the larvae after 12 h (Fig. 2A). The dead larvae exhibited no movement, no blood circulation, and a degenerated body (Fig. 2A). SVCV caused the death of more maoc1^–/– larvae than maoc1^+/+ larvae (Fig. 2B). Based on quantitative real-time PCR (qRT-PCR) assays, the expression of G, P, and N gene mRNA of SVCV monitoring SVCV replication was much higher in maoc1^−/− larvae than in maoc1^+/+ larvae (Fig. 2C to E).

FIG 2.

maoc1-null zebrafish larvae are more susceptible to SVCV infection than the WT zebrafish larvae. (A) Representative images of maoc1-null zebrafish larvae (4 dpf) and WT siblings (4 dpf) infected with or without SVCV for 12 h. The dead larvae exhibited no movement, no blood circulation, and a degenerated body. Dead larvae were identified by lack of movement, absence of blood circulation, and bodily degeneration (indicated by red arrows). (B) maoc1-null zebrafish (n = 96, 4 dpf) were more sensitive to SVCV infection than the WT (n = 96, 4 dpf) based on the survival ratio. SVCV viruses (~2.5 × 10⁷ TCID₅₀/mL) were added to maoc1-null and WT larvae, and the numbers of dead larvae were counted every hour from 0 to 24 h. (C to E) The virus replication number was greater in maoc1-null zebrafish larvae than in the WT zebrafish after infection with SVCV. maoc1-null larvae (maoc1^−/−) and the WT larvae (maoc1^+/+) are offspring of siblings. SVCV viruses (~2.5 × 10⁷ TCID₅₀/mL) were added to maoc1-null larvae (4 dpf) and the WT (4 dpf). After challenge for 12 h, the expression of G protein (C), P protein (D), and N protein (E) of SVCV was detected by qRT-PCR analysis. All data are presented as mean values based on three repeated experiments, and error bars indicate the ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Subsequently, we intraperitoneally (i.p.). injected SVCV, as well as using cell culture medium as a control, into maoc1^+/+ and maoc1^−/− adult zebrafish and then observed their phenotypes. Compared with SVCV-injected maoc1^+/+ adult zebrafish (3 months postfertilization [mpf]), SVCV-injected maoc1^−/− adult zebrafish (3 mpf) exhibited more swelling and hemorrhagic symptoms in the abdomen (Fig. 3A). We counted dead zebrafish at various time points after SVCV injection and created a survival curve. As shown in Fig. 3B, after challenge with SVCV, maoc1^+/+ zebrafish displayed a higher survival rate to than maoc1^−/− zebrafish. A histopathological assay indicated that maoc1^−/− zebrafish displayed necrosis and cytoplasmic vacuolization in liver compared with maoc1^+/+ zebrafish after SVCV infection (Fig. 3C). Moreover, qPCR assays for the expression of G, P, and N gene mRNA showed that SVCV replication was much higher in maoc1^−/− zebrafish liver than in maoc1^+/+ zebrafish liver (Fig. 3D to F).

FIG 3.

maoc1-null zebrafish adults are more sensitive to SVCV infection than WT zebrafish. (A) maoc1-null zebrafish (maoc1^−/−) (3 months postfertilization [3 mpf]) displayed more symptoms of abdominal hemorrhage after SVCV infection than WT zebrafish (maoc1^+/+) (3 mpf). The zebrafish were i.p. injected with either 10 μL cell culture medium or 10 μL SVCV (~2.5 × 10⁷ TCID₅₀/mL). The red arrows indicate hemorrhage regions. (B) maoc1-null zebrafish (3 mpf) (n = 20) were more sensitive to SVCV infection than the WT (3 mpf) (n = 20) based on the survival ratio. (C) Hematoxylin-eosin staining (H&E) of liver sections from maoc1-null zebrafish (maoc1^−/−) and WT zebrafish (maoc1^+/+) after i.p. injection with 10 μL cell culture medium or 10 μL SVCV (~2.5 × 10⁷ TCID₅₀/mL). Compared with WT zebrafish (maoc1^+/+), maoc1-null zebrafish (maoc1^−/−) displayed necrosis and cytoplasmic vacuolization in liver after SVCV infection. (D to F) The virus replication numbers were greater in maoc1-null zebrafish liver than in the WT after being infected with SVCV. After i.p. injection with 10 μL cell culture medium or 10 μL SVCV (~2.5 × 10⁷ TCID₅₀/mL) for 48 h, livers were dissected, and then the expression of G protein (D), P protein (E), and N protein (F) of SVCV was detected by qRT-PCR analysis. All data are presented as mean values based on three repeated experiments, and error bars indicate the ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Taken together, these data suggest that disruption of maoc1 in zebrafish facilitates virus propagation after infection with SVCV.

Zebrafish maoc1 inhibits virus replication in SVCV-infected EPC cells.

To further figure out the reason why the disruption of SVCV-induced maoc1 in zebrafish facilitates virus replication, we conducted a series of assays. Overexpression of maoc1 in epithelioma papulosum cyprini (EPC) cells caused a reduction of cytopathic effect (CPE) compared with overexpression of empty vector control after SVCV infection (Fig. 4A). Consistently, the titer of SVCV was significantly decreased in maoc1-overexpressed EPC cells compared with the control cells transfected with empty vector (Fig. 4B). Furthermore, SVCV replication indicated by the expression of G, P, and N gene mRNA was dramatically reduced in maoc1-overexpressed EPC cells compared with the control cells transfected with empty vector (Fig. 4C to E).

FIG 4.

Zebrafish maoc1 inhibits virus propagation in SVCV-infected EPC cells. (A) Overexpression of maoc1 enhanced cell survival of EPC cells after SVCV infection. EPC cells were transfected with 2 μg of HA-maoc1 or empty vector. At 24 h posttransfection, the cells were infected with SVCV (MOI of 0.1 to 10) at the dose indicated for 48 h. Then, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (B) Overexpression of maoc1 reduced virus titers in EPC cells after SVCV infection. The culture supernatant was collected from EPC cells infected with SVCV (MOI of 1), and the viral titer was measured by plaque assay. The results are representative of three independent experiments. (C to E) Overexpression of maoc1 reduced copy numbers of SVCV-related genes in SVCV-infected EPC cells. EPC cells were transfected with HA-maoc1 or empty vector and infected with SVCV (MOI of 1). After 24 h, total RNAs were extracted for examining the mRNA levels of the G protein (C), P protein (D), and N protein (E) of SVCV by qRT-PCR analysis. All data are presented as mean values based on three repeated experiments, and error bars indicate the ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

These data suggest that zebrafish maoc1 might play an inhibitory role in SVCV replication inside cells.

Zebrafish maoc1 inhibits SVCV-induced antiviral gene expression, but not poly (I:C)-induced antiviral gene expression.

It is well defined that some host factors can facilitate virus replication by negatively regulating antiviral innate immunity and subsequently suppressing antiviral gene expression (4–10). To determine the underlying mechanisms of how zebrafish maoc1 impacts SVCV replication, we examined the expression of typical antiviral genes between maoc1^−/− and maoc1^+/+ zebrafish after challenge with SVCV. As shown in Fig. 5A and B, unexpectedly, in both zebrafish larvae and adults, SVCV-induced expression of typical antiviral genes, such as ifn1, lta, and mxc, was much higher in maoc1^−/− than in maoc1^+/+ zebrafish (Fig. 5A and B). These results are well coordinated with the virus load in maoc1^−/− and maoc1^+/+ zebrafish after SVCV infection (Fig. 2 and 3) but contrast with the results of disrupting the negative regulator of antiviral innate immunity reported previously (4–10). They appeared to agree that more virus load in maoc1^−/− zebrafish induced higher expression of antiviral genes.

FIG 5.

Zebrafish maoc1 inhibits SVCV-induced, but not poly (I:C)-induced, key antiviral gene expression. (A) The induction of key antiviral genes, including ifn1, lta, and mxc, was decreased in maoc1-null larvae (maoc1^−/−) compared with the WT larvae (maoc1^+/+) after SVCV infection (~2.5 × 10⁷ TCID₅₀/mL) for 12 h. (B) The induction of key antiviral genes, including ifn1, lta, and mxc was increased in maoc1-null adults (maoc1^−/−) compared with the WT adults (maoc1^+/+) after i.p. injection with 10 μL cell culture medium or 10 μL SVCV (~2.5 × 10⁷ TCID₅₀/mL) for 48 h. (C) Overexpression of maoc1 did not suppress the expression of key antiviral genes, including ifn1, lta, and mxc, induced by treatment with poly (I:C) (1 μg/mL) in ZFL cells for 24 h. (D) Overexpression of maoc1 suppressed the expression of ifn, isg15, and viperin after SVCV (~2.5 × 10⁷ TCID₅₀/mL) infection in EPC cells. (E) Overexpression of maoc1 did not suppress the expression of ifn, isg15, and viperin induced by treatment with poly (I:C) (1 μg/mL) in EPC cells for 24 h. All data are presented as mean values based on three repeated experiments, and error bars indicate the ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

To further figure out the reason underlying this phenomenon, we used a viral RNA mimic, poly (I:C), instead of virus to activate the innate immune response and then examined the effect of maoc1 in this process. In zebrafish liver cells (ZFL) cells, transfection with poly (I:C) induced expression of antiviral genes ifn1, lta, and mxc as expected, but overexpression of maoc1 enhanced expression of ifn1 and had no effect on the expression of lta and mxc, inconsistent with the results obtained by SVCV challenge in maoc1^−/− and maoc1^+/+ zebrafish (Fig. 5C). In EPC cells, overexpression of maoc1 caused a significant reduction of SVCV-induced expression of typical antiviral genes, ifn, isg15, and viperin, in agreement with the results obtained by SVCV challenge in maoc1^−/− and maoc1^+/+ zebrafish larvae and adults (Fig. 5D). However, similar to ZFL cells transfected with poly (I:C), overexpression of maoc1 had no obvious effect on poly (I:C)-induced expression of ifn, isg15, and viperin (Fig. 5E). It was clear that zebrafish maoc1 did not affect antiviral gene expression but could directly repress SVCV replication, thus leading to the reduction of SVCV load in maoc1^+/+ zebrafish or maoc1-overexpressed cells.

Taken together, these data suggest that zebrafish do not affect the antiviral innate immune response but could directly suppress virus propagation.

Zebrafish maoc1 interacts with the phosphoprotein (P protein) of SVCV.

We performed further assays to determine the mechanism of zebrafish maoc1 in the attenuation of SVCV replication. The SVCV genome encodes 5 proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) (1, 9). In order to ascertain whether maoc1 can directly affect the factors of SVCV, we cloned these 5 genes into an expression vector and discovered that three of them, G, P, and N, could be readily expressed in cells as detected by Western blotting assay. Subsequently, we conducted coimmunoprecipitation assays to evaluate the potential interaction between maoc1 and G, P, or N, whereas the association between maoc1 with P protein was detected (Fig. 6A and B), but the association between maoc1 with G or N proteins was not (Fig. S3A and B). Furthermore, we confirmed the interaction of maoc1 and P protein during SVCV infection in EPC cells (Fig. 6C). Domain mapping showed that the C-terminal occludin domain of maoc1 was vital for binding to P protein (Fig. 6D), and the C-terminal domain of the P protein was critical for its association with maoc1 (Fig. 6E). Immunofluorescent staining further validated the predominant colocalization of maoc1 and P protein in the cytoplasm, but there was a trivial amount in the cell membrane (Fig. 6F).

FIG 6.

Zebrafish maoc1 interacts with SVCV phosphoprotein. (A and B) Flag-maoc1 associated with Myc-P. (C) Myc-maoc1 associated with SVCV phosphoprotein (P protein). (D) The occludin domain of maoc1 was required for binding to SVCV P protein. (E) The interaction between Maoc1 and P protein depended on the C domain of P protein. HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, total cell lysates were immunoprecipitated (IP) with anti-Flag or anti-Myc antibody-conjugated agarose beads. Then, the immunoprecipitants and cell lysates were detected with anti-Flag or anti-Myc antibody, respectively. (F) Maoc1 colocalizes with SVCV phosphoprotein (P protein). Green signals represent the overexpressed HA-P, red signals represent the overexpressed Flag-maoc1, and blue signals represent nuclei. Scale bar = 10 μm.

These data suggest that zebrafish maoc1 interacts with the P protein of SVCV to play its role in suppressing SVCV replication inside cells.

Zebrafish maoc1 induces autophagic degradation of SVCV phosphoprotein.

To determine the mechanism of how zebrafish maoc1 suppresses SVCV replication through interacting with P protein of SVCV, we examined the effect of maoc1 on the stability of P protein. Overexpression of maoc1 in HEK293T cells caused a reduction of overexpressed P protein in a dose-dependent manner but did not affect the N and G protein stability (Fig. 7A; Fig. S3C and D). Furthermore, in SVCV-infected EPC cells, overexpression of maoc1 caused the SVCV P protein to almost disappear (Fig. 7B). Treatment with cycloheximide (CHX), a protein synthesis inhibitor, did not block maoc1-induced P protein degradation, suggesting that new protein was not required for maoc1-mediated P protein degradation (Fig. 7C). In contrast, overexpression of P protein had no effect on maoc1 protein stability (Fig. 7D).

FIG 7.

maoc1 induces autophagic degradation of SVCV phosphoprotein. (A) maoc1 induced degradation of phosphoprotein (P) in a dose-dependent manner. HEK293T cells were transfected with Myc-empty, Myc-maoc1, and Flag-P for 24 h, and then the cells were harvested to perform immunoblotting. (B) maoc1 induced degradation of SVCV P protein in EPC cells after SVCV infection. EPC cells were transfected with Myc-empty or Myc-maoc1. After 24 h, SVCV (~2.5 × 10⁷ TCID₅₀/mL) was added in EPC cells for 24 h, and then the cells were harvested to detect SVCV P protein by anti-P antibody. (C) maoc1 accelerated degradation of P protein in the presence of cycloheximide (CHX). HEK293T cells were transfected with Myc-empty, Myc-maoc1, and Flag-P. After 18 h, CHX (50 ng/mL) was added to inhibit new protein synthesis, and the cells were harvested at different time points. (D) P protein did not alter the protein level of maoc1. HEK293T cells were transfected with Flag-empty, Flag-P, and Myc-maoc1 for 24 h, and then the cells were harvested to perform immunoblotting. (E) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-maoc1, and HA-P for 24 h and then cultured in the presence of the proteasome inhibitor, MG132 (20 μM), or DMSO for 12 h. (F) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-maoc1, and HA-P for 24 h and then cultured in the presence of the autophagy inhibitor, 3-MA (1 mM), or DMSO for 12 h. (G) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-maoc1, and HA-P for 24 h and then cultured in the presence of the lysosome inhibitor, NH₄Cl (5 mM), for 12 h. (H) maoc1 promoted the protein level of autophagy marker LC3-II. HEK293T cells were transfected with Myc-empty, Myc-maoc1, HA-empty, and HA-P and for 24 h, and then the cells were harvested to perform immunoblotting. (I) Treatment with 3-MA restored SVCV P protein degradation induced by maoc1 in EPC cells when infected with SVCV (~2.5 × 10⁷ TCID₅₀/mL). (J) Treatment with 3-MA relieved the suppressive role of maoc1 on SVCV propagation in EPC cells as revealed by SVCV titer determination.

To further determine what kind of protein degradation responds to maoc1-mediated P protein degradation, we utilized several well-known inhibitors, including the proteasomal inhibitor, MG132, the autophagy inhibitor, 3-methyladenine (3-MA), and the lysosomal proteolysis inhibitor, NH₄Cl. In addition, we examined the viability of inhibitor-treated cells and found that except for MG132, other inhibitors did not affect cell viability (Fig. S4). As shown in Fig. 7E, the addition of MG132 did not affect maoc1-induced P protein degradation. However, the addition of 3-MA restored the protein level of P protein obviously (Fig. 7F), which suggested that maoc1 might mediate autophagic degradation of P protein. As expected, the addition of NH₄Cl restored the protein level of P protein as well (Fig. 7G).

To further validate whether maoc1 could induce autophagic degradation of P protein, we examined the expression in cells of LC3, whose cleavage is a classic marker of autophagy (16). As shown in Fig. 7H, overexpression of maoc1 could indeed significantly enrich the LC3-II level, and this enrichment was further amplified when P protein was coexpressed (Fig. 7H). In agreement, knockdown of maoc1 led to a decrease of GFP-lc3 aggregation after SVCV infection in ZFL cells (Fig. S5A). The efficiency of maoc1-targeting small interfering RNA (siRNA)-mediated knockdown was evaluated by qPCR and Western blotting (Fig. S5B and C). This suggested that maoc1 could induce autophagy, which might account for P protein degradation.

Furthermore, addition of 3-MA restored maoc1-induced degradation of P protein in SVCV-infected EPC cells (Fig. 7I). Consistently, addition of 3-MA also restored SVCV titer in maoc1-overexpressed EPC cells (Fig. 7J).

Collectively, these data suggest that zebrafish maoc1 induces autophagy-lysosome-dependent degradation of SVCV phosphoprotein to suppress SVCV proliferation.

Zebrafish maoc1 inhibits dimerization of SVCV phosphoprotein to eliminate its promoted immune evasion.

It was reported previously that the P protein of SVCV inhibits the cellular IFN response to facilitate virus replication, in which the P protein promotes immune evasion by its dimerization (17). To determine whether zebrafish maoc1 could affect the dimerization of P protein, we performed a coimmunoprecipitation (co-IP) assay. As shown in Fig. 8A, coexpression of maoc1 reduced dimerization of P protein obviously. We then investigated whether zebrafish maoc1 could affect P protein-mediated immune escape by detecting typical antiviral gene expression, including ifn1, lta, and mxc in ZFL cells. Overexpression of P protein suppressed poly(I:C)-stimulated expression of ifn1, lta, and mxc significantly (Fig. 8B to D). However, coexpression of maoc1 restored the suppressive role of P protein on poly (I:C)-stimulated expression of ifn1, lta, and mxc dramatically (Fig. 8B to D). These data suggest that zebrafish maoc1 could eliminate immune evasion by preventing the dimerization of P protein.

FIG 8.

maoc1 inhibits dimerization of SVCV phosphoprotein dimerization and P-induced immune escape. (A) maoc1 inhibited dimerization of phosphoprotein (P protein). HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, total cell lysates were immunoprecipitated (IP) with anti-HA agarose beads. Then, the immunoprecipitants and cell lysates were detected with anti-Myc or anti-Flag antibody, respectively. (B to D) maoc1 restored the repressive role of P protein on the expression of key antiviral genes, including ifn1 (B), lta (C), and mxc (D), induced by treatment with poly (I:C). ZFL cells seeded in 100-mm dishes were transfected with the indicated plasmids. After 24 h, the cells were transfected with poly (I:C) (1 μg/mL) for 24 h, and then qRT-PCR was performed.

DISCUSSION

In order to fight against virus infection, hosts develop multiple defense mechanisms to eliminate viruses or limit virus replication (18). Recently, it has gotten much attention that the host factors affect virus replication by directly acting on viral factors (12–14). In this study, we identify that zebrafish maoc1 is induced by SVCV infection and directly mediates autophagic degradation of P protein to interrupt SVCV propagation, revealing a unique defense mechanism of zebrafish against virus infection.

Intriguingly, the occludin domain identified in maoc1 exists in several proteins, such as OCLN, ELLs (ELL, ELL2, ELL3), OCEL1, and MARVELD2 (19, 20). Among them, OCLN is found to promote propagation of different viruses, including HCV, human immunodeficiency virus (HIV), and porcine epidemic diarrhea virus (PEDV) (21–25), in contrast to maoc1. It is still elusive whether other occludin family members are also involved in virus replication. Notably, it has been revealed that other negative-strand RNA viruses (NSVs) also contain P-like proteins, which exhibit similar function in the modulation of innate immune response as that of SVCV P protein (2, 26–31). Here, we find that the occludin domain of maoc1 is required for binding to P protein and mediating subsequent degradation of P protein, which can affect virus propagation. Is it possible that other occludin family proteins are also involved in affecting NSV propagation through their occludin domain binding to P protein of viruses? Further investigation of this question will shed new light on the depiction of host defense mechanisms in response to virus invasion.

Directly observing the process of maoc1 interrupting SVCV replication and propagation will give strong evidence to support the role of maoc1 in the attenuation of SVCV propagation. However, due to the lack of tools for real-time monitoring of SVCV replication in cells, in this study, we cannot provide evidence to support that maoc1 directly interrupts SVCV replication.

Autophagy is an evolutionarily conserved degradation mechanism that disassembles dysfunctional cellular components and therefore facilitates the capture and clearance of invading pathogens (32, 33). In addition, autophagy is also an active host defense strategy to eliminate invading viruses at the early stages of viral infection through limiting the replication and proliferation potential of viruses (34, 35). On the other hand, viruses have evolved the ability to hijack autophagy-related proteins to weaken the degree of autophagy or use the autophagy machinery to target the host defense proteins involved in their immune response and thereby promote virus proliferation (34, 36). This study shows that maoc1 causes P protein degradation via the autophagy-lysosome pathway, leading to the inhibition of SVCV replication. In addition, maoc1 is induced by SVCV infection. Thus, these findings uncover an effective defense mechanism of the host against virus infection by utilizing cellular autophagy machinery.

Of note, SVC disease causes tremendous loss in the aquaculture industry (2). However, to date, there is still no operational strategy for the treatment of this disease. Demonstrating the genetic basis of fish for defending against SVCV infection will help to cultivate SVCV-resistant fish strains, benefiting the aquaculture industry in the future.

MATERIALS AND METHODS

Cell culture and transfection.

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS). Zebrafish liver cells (ZFL) were grown in 50% L-15 (Invitrogen), 35% DMEM-HG (Invitrogen), and 15% Ham F12 medium (Invitrogen) supplemented with 0.15 g/L sodium bicarbonate (Sigma-Aldrich), 15 mM HEPES (Sigma-Aldrich), and 10% FBS. Epithelioma papulosum cyprini (EPC) cells were maintained in medium 199(M-199) (Invitrogen) containing 10% FBS. HEK 293T cells were maintained at 37°C, and ZFL and EPC cells were maintained at 28°C. All cell lines were cultured in a humidified incubator containing 5% CO2, verified to be free of Mycoplasma contamination before use. VigoFect (Vigorous Biotechnology, Beijing, China) and Fish Trans (MST, FT2020, Wuhan, China) were used for cell transfection.

Zebrafish.

Zebrafish (Danio rerio) strain AB fish were raised, maintained, and staged according to standard protocols. We used CRISPR/Cas9 to knockout maoc1 in zebrafish. First, maoc1 single guide RNA (sgRNA) was designed using the CRISPR design tool (http://crispr.mit.edu). The sgRNA sequence was 5′-GGAGAACGACAGATTTTAAC-3′. The zebrafish codon optimized Cas9 plasmid was digested with XbaI and then purified and transcribed using the T7 mMessage machine kit (Ambion). PUC9 guide RNA (gRNA) vector was used to amplify maoc1 sgRNA template. sgRNA was synthesized using the Transcript Aid T7 high-yield transcription kit (Fermentas). Zebrafish embryos at the one-cell stage were injected with 1 ng Cas9 RNA and 0.15 ng sgRNA per embryo. The mutations were initially detected using a heteroduplex mobility assay (HMA) as previously described (37). If the HMA results were positive, the remaining embryos were raised to adulthood as the F0 generation and were then backcrossed with WT zebrafish (strain AB) to generate the F1 generation. F1s were genotyped with HMAs. Genotypes were confirmed by sequencing target sites. Heterozygous F1s were back-crossed with WT zebrafish (strain AB; disallowing offspring-parent mating) to generate the F2 generation. F2 adults carrying the target mutation were intercrossed to generate F3 offspring. The F3 generation contained WT (+/+), heterozygous (+/−), and homozygous (−/−) individuals. The primers used to identify mutants were as follows: forward primer, 5′-CAGACTCTGGAATCCACATC-3′; reverse primer, 5′-TATTACATTAGTCGCTACTGG′. The novel mutants were named following zebrafish nomenclature guidelines: maoc1 ihbsynm1/ihbsynm1 (https://zfin.org/ZDB-ALT-220726-1). All zebrafish procedures were performed approval of the Institutional Animal Care and Use Committee (IACUC) at the Institute of Hydrobiology, Chinese Academy of Sciences, and in compliance with IACUC guidelines.

Plasmid construction and reagents.

Zebrafish gene maoc1 (XM_686442.7) and its truncated mutants were PCR amplified from zebrafish cDNA using PCR. Amplified genes were subcloned into pCMV-Myc (Clontech), pCMV-HA (Clontech), or pCMV-Flag (Clontech) vectors. All constructs were verified by DNA sequencing. These plasmids, including Flag-P, Flag-N, and Flag-G, were obtained from Shun Li (Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China). Poly (I:C) was purchased from InvivoGen (San Diego, CA) and transfected with Lipofectamine 2000 (Thermo Fisher Scientific) at a final concentration of 1 mg/mL. Cycloheximide (CHX), MG132, and 3-MA were purchased from MedChemExpress. NH4Cl was purchased from Sigma-Aldrich. The antibodies used were as follows: anti-Myc antibody (Ab) (9E10; Santa Cruz Biotechnology), anti-Flag Ab (F1804-5MG; Sigma-Aldrich), anti-hemagglutinin (HA) Ab (MMS-101R; Covance), anti-β-actin Ab (AC026; ABclonal), anti-SVCV P Ab (Shun Li lab), and anti-LC3 Ab (ab48394; Abcam).

Virus infection and plaque assay.

Spring viremia of carp virus strain OMG067 was propagated in EPC cells until the cytopathic effect (CPE) was complete, and the culture medium was collected and stored at −80°C until use. For virus titration, EPC cells were cultured in 96-well plates. The culture supernatant (containing virus) was diluted in serial dilutions (10⁻¹ to 10⁻¹¹) in sterile 1.5-mL tubes using M-199 medium. Subsequently, the diluted viruses were added to EPC cells seeded into 96-well plates. After 4 days, the plates were observed under a microscope, and the area of detached cells after infection with virus >50% in one well were counted as positive. The titers for SVCV infection were calculated using the Spearman-Kärber method and represented as 50% tissue culture-infective dose (TCID₅₀) assay. The experiments were repeated three times for statistical analysis.

For viral infection of zebrafish larvae, 50 larvae (4 dpf) were pooled in a disposable 60-mm cell culture dish filled with 5 mL of egg water and 1 mL of SVCV (~2.5 × 10⁷ TCID₅₀/mL) solution. All infections were performed at 28°C for 12 h at least three times for statistic assays. For examining gene expression, the total RNA was extracted, and quantitative real-time PCR (qRT-PCR) assays were conducted. For survival ratio assays, zebrafish larvae were placed in 96-well plates individually, and then 100 μL of egg water containing SVCV (5 mL of egg water plus 2 mL of SVCV (~2.5 × 10⁷ TCID₅₀/μL) was added to each well). The mortality was monitored every 1 h over a 24-h period.

For viral injection of adult zebrafish, 3 mpf-zebrafish were intraperitoneally (i.p.) injected with SVCV (~2.5 × 10⁷ TCID₅₀/mL) at 10 μL per individual. Zebrafish i.p. injection with M-199 cell culture medium was used as the control. After viral challenge for 48 h, zebrafish were anesthetized with tricaine methane sulfonate and dissected. The livers were collected and stored at −80°C for further qPCR assays.

For the plaque assay, EPC cells were transfected with 2 μg of HA-tagged maoc1 or HA-empty vector. After 24 h, cells were infected with SVCV for 48 h at the dose indicated. Subsequently, the supernatant was collected from EPC cells infected with SVCV (multiplicity of infection [MOI] of 1) for the detection of virus titers, and the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with 1% crystal violet for visualizing CPE.

Quantitative real-time PCR (qRT-PCR) analysis.

Total RNA was extracted from cells, embryos (n = 50), or tissues (n = 3) using the RNAiso Plus kit (TaKaRa Bio, Beijing, China), following the manufacturer’s instructions. cDNAs were synthesized using the RevertAid first-strand cDNA synthesis kit (Thermo Scientific, Waltham, MA, USA). MonAmpTM SYBR green qPCR mix (high Rox) (Monad Bio, Shanghai, China) was used for qRT-PCR assays. qRT-PCR was performed with three biological replicates, and each experiment was repeated at least three times independently. The primers for qPCR assays are listed in Table S1.

Coimmunoprecipitation assay and Western blot analysis.

Anti-Flag antibody and anti-HA antibody-conjugated agarose beads were purchased from Sigma. For Western blot analysis and coimmunoprecipitation of overexpressed proteins, the experimental procedures have been described previously (6). The Fuji Film LAS4000 mini luminescent image analyzer was used for photographing the blots. Multigauge V3.0 was used for quantifying the protein levels based on the band density obtained in the Western blot analysis.

Immunofluorescence confocal microscopy.

Cells grown on glass coverslips were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin. Then, the cells were stained with the indicated primary antibodies followed by incubation with fluorescent-dye-conjugated secondary antibodies. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich). Imaging of the cells was carried out using a Leica laser-scanning confocal microscope.

Hematoxylin and eosin (H&E) staining.

Livers from the controls or virus-infected zebrafish were dissected, fixed with 4% formaldehyde (PFA) overnight at room temperature, and then embedded into paraffin, sectioned, stained with hematoxylin and eosin solution, and examined by light microscopy for histological changes.

Cell viability assays.

Cell viability was assessed with a Cell Counting Kit-8 (CCK-8) assay kit (Eallbio). HEK 293T and EPC cells were cultured in 96-well plates overnight, and then dimethyl sulfoxide (DMSO), MG132, 3-MA, and NH₄Cl were added according to the previously mentioned concentrations. After 12 h, 10 μL CCK-8 reagent was added into each well for 3 h, and absorbance readings at 450 nm were obtained using a spectrophotometric plate reader. The data were obtained from the measurement of 3 replicate wells for each data point. The percentage at each concentration relative to the control was presented as cell viability.

siRNA treatment.

The maoc1-targeting siRNA was obtained from GenePharma (Suzhou, China). The sequences were as follows: si-maoc1 sense, GCAAUACAAAGCCGUCUUUTT; si-maoc1 antisense, AAAGACGGCUUUGUAUUGCTT. According to the manufacturer’s instructions, Lipofectamine 3000 transfection reagent (Thermo Fisher) was applied for transient transfection of ZFL cells. The efficiency of siRNA-mediated knockdown after treatment was evaluated by qPCR and Western blotting.

Statistical analysis.

The statistical analysis was performed using GraphPad Prism version 8.0 (unpaired t tests). Data are presented as the mean ± standard error of the mean (SEM) of three biological replicates. Each experiment was repeated at least three times independently. For zebrafish survival analysis, the Kaplan-Meier method was adopted to generate graphs, and the survival curves were analyzed by log-rank analysis. A P value of <0.05 was considered significant. Statistical significance is represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.