ABSTRACT
Interferon (IFN) production activated by phosphorylated interferon regulatory factor 7 (IRF7) is a pivotal process during host antiviral infection. For viruses, suppressing the host IFN response is beneficial for viral proliferation; in such cases, evoking host-derived IFN negative regulators would be very useful for viruses. Here, we report that the zebrafish rapunzel 5 (RPZ5) protein which activated by virus degraded phosphorylated IRF7 is activated by TANK-binding kinase 1 (TBK1), leading to a reduction in IFN production. Upon viral infection, zebrafish rpz5 was significantly upregulated, as was ifn, in response to the stimulation. Overexpression of RPZ5 blunted the IFN expression induced by both viral and retinoic acid-inducible gene I (RIG-I) like-receptor (RLR) factors. Subsequently, RPZ5 interacted with RLRs but did not affect the stabilization of the proteins in the normal state. Interestingly, RPZ5 degraded the phosphorylated IRF7 under TBK1 activation through K48-linked ubiquitination. Finally, the overexpression of RPZ5 remarkably reduced the host cell antiviral capacity. These findings suggest that zebrafish RPZ5 is a negative regulator of phosphorylated IRF7 and attenuates IFN expression during viral infection, providing insight into the IFN balance mechanism in fish.
IMPORTANCE The phosphorylation of IRF7 is helpful for host IFN production to defend against viral infection; thus, it is a potential target for viruses to mitigate the antiviral response. We report that the fish RPZ5 is an IFN negative regulator induced by fish viruses and degrades the phosphorylated IRF7 activated by TBK1, leading to IFN suppression and promotion of viral proliferation. These findings reveal a novel mechanism for interactions between the host cell and viruses in the lower vertebrate.
INTRODUCTION
Upon viral infection, virus-specific pathogen-associated molecular patterns (PAMPs) are recognized by host pathogen-recognition receptors (PRRs), such as Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and NOD-like receptors (NLRs) (1, 2). RLRs, including RIG-I and melanoma differentiation-associated gene 5 (MDA5) (3, 4), go through conformational changes and interact with the mitochondrial antiviral signaling protein (MAVS, also known as VISA, IPS-1, and Cardiff) when infected with viruses (5–8). Subsequently, activated MAVS recruits a mediator of interferon (IFN) regulatory factor 3 (IRF3) activation (MITA, also known as STING, ERIS, and MPYS) (9–12) and TANK-binding kinase 1 (TBK1), leading to phosphorylation, dimerization, and nuclear accumulation of IRF3/7 and ultimately inducing the expression of IFN and inflammatory cytokines (13, 14). Although IRF3 seems to play an important role, as it is expressed ubiquitously and constitutively and is involved in early responses to viral infection, IRF7 is also expressed in most cell types, is upregulated by viral infections, and functions as a crucial transcription factor to activate IFN expression to defend against viral influence (15, 16). Studies with knockout mice have revealed that IRF7 plays a dominant role in the regulation of IFN induction (13, 17).
Since IFN plays a critical role in host immune defenses against viral infection, it is always antagonized by viruses for the promotion of viral proliferation. Several fish proteins have also been identified as negative regulators that target the key molecules in RLR signaling to block IFN production during the process of viral infection. For example, zebrafish PIAS4a is significantly upregulated by polyinosinic-poly(C) [poly(I·C)] and acts as a negative regulator in MAVS-mediated signaling cascades (18). Additionally, zebrafish IRF10 is induced by poly(I·C) and inhibits MITA-mediated IFN induction (19), and the alternatively spliced isoforms of zebrafish TBK1 suppress IFN expression in response to virus infection by inhibiting TBK1-IRF3 interactions and IRF3 phosphorylation (20). Moreover, zebrafish MAVS_tv2 or NDRG1a is induced upon virus infection and functions as a negative regulator in IRF7-mediated IFN production (21, 22), and zebrafish Forkhead box O (FOXO) 3b is stimulated by virus infection and represses the IFN response by impeding the transactivity of IRF3 and IRF7 (23). More fish IFN negative regulators are being discovered, indicating that an enormous and complex IFN response balance system also exists in fish.
Rapunzel 5 (RPZ5), which is absent in mammals, belongs to the rpz family, which is composed of rpz, rpz2, rpz3, and rpz4 in zebrafish. A previous study showed that the zebrafish mutant rapunzel, which results from a missense mutation in the rpz gene, has heterozygous defects in bone development, resulting in skeletal overgrowth (24). rpz is a founding member of a novel gene family, but its function remains unclear. RPZ5 has two splice variants; the long transcript encodes a 45-kDa protein, and the short transcript is 3′ incomplete. To date, there is little evidence to indicate the involvement of RPZ5 in the IFN response. In this study, we report that zebrafish RPZ5 (the long transcript) is a potent negative regulator of RLR-mediated IFN production. Mechanistically, RPZ5 is associated with IRF7 and promotes the K48-linked ubiquitination and degradation of phosphorylated IRF7, which results in the inhibition of IFN expression and the cellular antiviral response. This information indicates a novel role of fish RPZ5 in IFN production and may provide new insight into the function of the rpz family.
RESULTS
Induction patterns of zebrafish rpz5 by SVCV infection.As TBK1 is a crucial kinase in the IFN response, we attempted to unambiguously identify TBK1-associated proteins through coimmunoprecipitation (Co-IP) screening. Epithelioma papulosum cyprini (EPC) cells were transfected with Flag-tagged TBK1 or an empty vector, and the TBK1-associated complexes were immunoprecipitated using anti-Flag-agarose beads. Further mass spectrum analysis and BLAST searches of the GenBank databases revealed that RPZ5 was one of the proteins specifically associated with TBK1. In zebrafish, spring viremia of carp virus (SVCV) is an efficient pathogen that elicits host IFN expression, and poly(I·C) mimics viral RNA, significantly inducing IFN production. The expression patterns of rpz5 transcripts were monitored in this system in vitro. As shown in Fig. 1A, after transfection with poly(I·C), the expression level of rpz5 was upregulated and reached a peak at 24 h. ifnφ1 and irf7 (an antiviral gene and IFN regulator, respectively [25, 26]) were also upregulated (Fig. 1B and C). Subsequent to infection with SVCV, the mRNA level of rpz5 increased as early as 6 to 12 h later, decreased at 24 h, and increased again at 48 h (Fig. 1D). Similarly, the induction of ifnφ1 and irf7 expression was also observed in zebrafish liver (ZFL) cells infected with SVCV (Fig. 1E and F). These results suggest that zebrafish RPZ5 expression is induced upon virus infection and thus is likely involved in the host antiviral response.
RPZ5 is stimulated by virus infection. (A to F) qPCR detection of the transcriptional levels of rpz5, ifnφ1, and irf7 on stimulation. ZFL cells seeded on 6-well plates overnight and transfected with poly(I·C) (2 μg/ml) (A to C) or SVCV (MOI, 1) (D to F). At the time points 6, 12, 24, and 48 h, total RNA was extracted for further qPCR assays. The β-actin gene was used as an internal control for normalization. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from the control (*, P < 0.05). Experiments were repeated at least three times with similar results.
RPZ5 inhibits SVCV or poly(I·C)-stimulated IFN activation.RPZ5 may be related to the host response to viral infection, and IFN is a critical cytokine in the host antiviral response. Therefore, the role of RPZ5 in IFN expression was subsequently monitored. As shown in Fig. 2A to C, when stimulated with poly(I·C), IFNφ1pro activation was significantly induced in the control group, whereas this induction was significantly inhibited by the overexpression of RPZ5 in a dose-dependent manner. RPZ5 also suppressed SVCV-induced IFNφ1pro activity in a dose-dependent manner. In the host IFN response, the ISRE motif is considered the binding site of IFN-stimulated genes (ISGs) responding to the transcriptional factors. After cotransfection with RPZ5 and ISRE-Luc and stimulation with poly(I·C) or SVCV, the activation of ISRE decreased in a dose-dependent manner in the RPZ5 group compared to that in the control group (Fig. 2D to F). Moreover, the results from the quantitative PCR (qPCR) experiments indicated that the overexpression of RPZ5 in EPC cells impaired poly(I·C)-induced expression of downstream genes such as ifn and vig1 (Fig. 2G to L). Next, we investigated the function of endogenous RPZ5 in poly(I·C)-induced IFN production. We used three RPZ5-specific small interfering RNAs (siRNAs) in this study. Compared to siRPZ5#2, siRPZ5#1 significantly inhibited the expression of RPZ5 (Fig. 2M). In qPCR assays, the poly(I·C)-triggered expression of ifn was increased by RPZ5 knockdown using siRPZ5#1 (Fig. 2N). Similarly, the knockdown of RPZ5 also potentiated the poly(I·C)-induced transcription of vig1 (Fig. 2O). Collectively, these data demonstrate that RPZ5 blocks SVCV and poly(I·C)-induced IFN expression.
Inhibition of IFNφ1 by overexpression of RPZ5. (A to F) Overexpression of RPZ5 suppresses SVCV/poly(I·C)-induced IFNφ1pro and ISRE activation. EPC cells were seeded in 24-well plates and transfected the next day with 250 ng IFNφ1pro-Luc or ISRE-Luc and 25 ng pRL-TK, plus 200 ng Myc-RPZ5 or 400 ng Myc-RPZ5 or pCMV-Myc (control vector). At 24 h posttransfection, cells were untreated (null) or treated with poly(I·C) (1 μg/ml) or SVCV (MOI, 1) for 24 h. pRL-TK was used as a control. The cells were collected for calculating the cell survival rate (A and D) and then lysed for IB (B and E) and luciferase assay (C and F). (G to L) Overexpression of RPZ5 inhibits the expression of ifn and vig1 induced by poly(I·C) in EPC cells. EPC cells seeded in 6-well plates overnight were transfected with 2 μg Myc-RPZ5 or empty vector and transfected with poly(I·C) (1 μg/ml) at 24 h posttransfection. At 24 h after stimulation, the cells were collected to calculate the cell survival rate (G and J) and then lysed for IB (H and K) and qPCR analysis (I and L). The β-actin gene was used as an internal control for normalization. (M) Effects of RPZ5 RNA interference (RNAi) on the expression of endogenous RPZ5. EPC cells were seeded in 6-well plates overnight and transfected with 100 nM siRPZ5 #1, siRPZ5#2, or siNC. At 24 h posttransfection, the cells were transfected with poly(I·C). At 24 h poststimulation, total RNA was extracted to examine the transcriptional levels of RPZ5. (N and O) Effects of RPZ5 RNAi on the poly(I·C)-induced ifn and vig1 transcription. EPC cells were seeded in 6-well plates and transfected with 100 nM siNC or siRPZ5#2. At 24 h posttransfection, cells were untreated or transfected with poly(I·C) for 24 h before qPCR analysis was performed. The relative transcriptional levels were normalized to the transcription of the β-actin gene and represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from the control (*, P < 0.05). Experiments were repeated at least three times with similar results. lucif. act., luciferase activity.
RPZ5 blocks RLR-mediated IFN induction.The fish RLR signaling cascade has been reported to be a pivotal pathway for the activation of IFN expression (19). Thus, to determine whether RPZ5 participates in the IFN activation mediated by RLR factors, cells were cotransfected with RPZ5 and RLR molecules. As shown in Fig. 3A, overexpression of key RLR molecules led to a significant induction of IFNφ1pro activity, and such activation was repressed by the overexpression of RPZ5. Similarly, the induction of ISRE by the RLRs was also blunted by the overexpression of RPZ5 (Fig. 3B). These data suggest that RPZ5 negatively regulates IFN production activated by RLR cascades.
RPZ5 represses the activation of IFNφ1 and ISRE induced by RLRs. (A and B) Overexpression of RPZ5 inhibits the activation of IFNφ1/ISRE induced by RLRs. EPC cells were cotransfected with MAVS, TBK, MITA, IRF3, or IRF7 and pcDNA3.1-RPZ5 or pcDNA3.1(+) plus IFNφ1pro-Luc (A) or ISRE-Luc (B) at a 1:1:1 ratio. pRL-TK was used as a control. At 24 h posttransfection, cells were collected for the detection of luciferase activities. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from the control (*, P < 0.05). Experiments were repeated at least three times with similar results.
RPZ5 associates with the RLR axis and is located in the cytoplasm.Given that RPZ5 attenuates IFN expression activated by RLR molecules, whether RPZ5 interacts with the RLRs at the protein level was investigated. HEK 293T cells were cotransfected with Myc-RPZ5 and Flag-tagged RLR factors, including MAVS, MITA, TBK1, IRF3, and IRF7. The results showed that most of the anti-Flag antibody (Ab)-immunoprecipitated protein complexes containing RLR proteins were recognized by the anti-Myc Ab, suggesting that RPZ5 associates with the RLR proteins MAVS, MITA, TBK1, IRF3, and IRF7 (Fig. 4A). In addition, the subcellular locations of RPZ5 were monitored, and confocal microscopy analysis revealed that the RPZ5-enhanced green fluorescent protein (RPZ5-EGFP) signal was mainly distributed in the cytoplasm (Fig. 4B). Furthermore, as shown in Fig. 4C and D, the red signals from TBK1 or IRF7 that were uniformly distributed in the cytoplasm almost overlapped the green signal from RPZ5. In contrast, cells transfected with EGFP-RPZ5 and pDsRed-ER or pDsRed-Golgi did not yield an overlapping image (Fig. 4E and F). Moreover, the green fluorescent signals of RPZ5 were detected only in the cytosol with or without poly(I·C) stimulation. Taken together, these data suggest that RPZ5 is located in the cytoplasm and associates with RLR factors.
RPZ5 associates with RLR axis. (A) HEK 293T cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 μg each). After 24 h, cell lysates were immunoprecipitated (IP) with anti-Flag affinity gels. Then, the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-Flag Abs, respectively. (B to F) RPZ5 localizes in the cytoplasm. EPC cells were plated onto coverslips in 6-well plates and cotransfected with 1 μg RPZ5-EGFP and 1 μg the empty vector (B), TBK1-DsRed (C), IRF7-DsRed (D), ER-DsRed (E), or Golgi-DsRed (F), respectively. After 24 h, the cells were fixed and subjected for confocal microscopy analysis. Green signals represent the overexpressed RPZ5, and blue staining indicates nucleus region (original magnification ×63; oil immersion objective). (G) EPC cells seeded onto microscopy cover glass in 6-well plates were transfected with 2 μg EGFP-RPZ5 or the empty vector. After 24 h, the cells were untreated (null) or transfected with 2 μg poly(I·C) for 24 h; then, the cells were fixed and subjected to confocal microscopy analysis. Green signals represent the RPZ5 protein signal, and blue staining indicates the nucleus region (original magnification, ×63; oil immersion objective). Scale bar = 10 μm. All experiments were repeated at least three times with similar results.
RPZ5 degrades TBK1-mediated phosphorylation of IRF7.To investigate the regulatory mechanism of RPZ5 on the RLR axis, we examined the effect of RPZ5 on RLR molecules at the protein level. MAVS-, TBK1-, MITA-, IRF3-, and IRF7-Myc expression vectors were cotransfected with hemagglutinin (HA)-RPZ5 or an empty vector. As shown in Fig. 5A, overexpressed RPZ5 did not cause an obvious change in RLR molecules at the protein level. Given that IRF3/7 phosphorylation is indispensable for mediating the IFN response, whether the phosphorylation of IRF3/7 is influenced by RPZ5 needs to be clarified. As shown in Fig. 5B and C, cotransfection with Flag-TBK1 caused a shift of IRF7 to higher-molecular-weight bands, and the amount of IRF7 was dramatically reduced by the overexpression of RPZ5 in a dose-dependent manner. In contrast, the phosphorylation of IRF3 caused by TBK1 did not change with the overexpression of RPZ5. Accordingly, to characterize the functional domain of IRF7 regulated by RPZ5, two truncated mutants of IRF7 were constructed, IRF7-ΔN (lacking the DNA-binding domain [DBD]) and IRF7-ΔC (lacking the IRF-association domain [IAD]) (Fig. 5D). As shown in Fig. 5E, consistent with the wild-type IRF7, IRF7-ΔN bound to RPZ5, but such an association was abrogated in the IRF7-ΔC group. In addition, domain mapping analysis of RPZ5 indicated that the C-terminal domain of RPZ5 (amino acids [aa] 99 to 393) was required for RPZ5 and IRF7 association (Fig. 5F). The abundance of the IRF7-ΔC mutant also decreased when cotransfected with TBK1 and RPZ5, suggesting that the N-terminal DBD of IRF7 is the target of RPZ5 for degradation (Fig. 5G). Collectively, these data suggest that RPZ5 specifically promotes the degradation of phosphorylated IRF7, and the target region is the DBD.
RPZ5 degrades phosphorylated IRF7. (A) RPZ5 has no effect on the exogenous RLR factors. EPC cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (1 μg each) for 24 h. The cell lysates were subjected to IB with anti-Myc, anti-HA, and anti-β-actin Abs. (B and C) Overexpression of RPZ5 degrades phosphorylated IRF7 in a dose-dependent manner. HEK 293T cells were seeded in 6-well plates overnight and cotransfected with 1 μg Flag-TBK1 and 1 μg empty vector, and Myc-RPZ5 (B) or Myc-RPZ5 (0.5, 1.5, or 2.5 μg) (C), together with 1 μg HA-IRF7/IRF3 for 24 h. Then, the whole-cell lysates were subjected to IB with the anti-HA, anti-Myc, anti-Flag, and anti-β-actin Abs. (D) Schematic representation of wild-type IRF7 and two mutants (IRF7-ΔN lacking the N-terminal DBD domain and IRF7-ΔC lacking the C-terminal IAD domain). (E) RPZ5 interacts with IRF7 via its IAD domain. HEK 293T cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 μg each). After 24 h, cell lysates were IP with anti-Myc-affinity gel. Then, the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-HA Abs, respectively. (F) IRF7 interacts with RPZ5 via its C terminus. Shown is a schematic representation of wild-type RPZ5 and two mutants. HEK 293T cells seeded in 10-cm2 dishes were transfected with the indicated plasmids (5 μg each). After 24 h, cell lysates were IP with anti-Myc-affinity gel. Then, the immunoprecipitates and cell lysates were analyzed by IB with the anti-Myc and anti-HA Abs, respectively. (G) RPZ5 targets the DBD domain of IRF7 for degradation. HEK 293T cells were seeded in 6-well plates overnight and transfected with the indicated plasmids (1 μg each) for 24 h. Then, the lysates were detected by IB with the anti-HA, anti-Myc, anti-Flag, and anti-β-actin Abs. All experiments were repeated at least three times with similar results.
RPZ5 mediates the ubiquitination of phosphorylated IRF7.To probe the mechanisms responsible for the degradation of IRF7 by RPZ5, cells were treated with MG132, which is a proteasome inhibitor, and the abundance of phosphorylated IRF7 in the presence or absence of RPZ5 was examined. As shown in Fig. 6A, RPZ5 caused the degradation of phosphorylated IRF7, which could be rescued by increasing the concentration of MG132, indicating that RPZ5 degrades phosphorylated IRF7 in a proteasome-dependent manner. To further clarify whether phosphorylated IRF7 was degraded by RPZ5 due to ubiquitination, Myc-IRF7, HA-RPZ5, Flag-TBK1, and HA-ubiquitin (HA-Ub) were cotransfected in the presence or absence of MG132. Following the immunoprecipitation of Myc-IRF7, immunoblot analysis revealed that RPZ5 potentiated the ubiquitination of phosphorylated IRF7 (Fig. 6B). K48 and K63, the lysines at positions 48 and 63 of ubiquitin linked with polyubiquitin chains, respectively, are two canonical polyubiquitin chain linkages. Given that K48-linked polyubiquitin chain modification leads to the targeting of proteins for proteasome recognition and degradation, whereas K63-linked polyubiquitin chain modification enhances the stability of the target proteins (27, 28), we chose to investigate whether RPZ5 promoted the K48- or K63-linked ubiquitination of phosphorylated IRF7. Thus, cells were cotransfected with Myc-IRF7, HA-RPZ5, Flag-TBK1, and HA-Ub, HA-Ub-K48O, or HA-Ub-K63O. As shown in Fig. 6C, RPZ5 promoted K48-linked ubiquitination of phosphorylated IRF7 but not K63-linked ubiquitination. These results demonstrate that RPZ5 induces the K48-linked Ub-proteasomal degradation of phosphorylated IRF7.
RPZ5 mediates ubiquitination of phosphorylated IRF7. (A) RPZ5 induces phosphorylated IRF7 degradation and is rescued by MG132 in a dose-dependent manner. HEK 293T cells were seeded in 6-well plates overnight and transfected with 1 μg HA-IRF7, 1 μg Flag-TBK1, and 1 μg Myc-RPZ5 or empty vector. At 18 h posttransfection, the cells were treated with dimethyl sulfoxide (DMSO) or MG132 (10, 20, or 40 μM) for 6 h. Then, the cells were harvested for IB with the Abs indicated. (B) RPZ5 promotes the ubiquitination of phosphorylated IRF7. HEK 293T cells were transfected with 4 μg Myc-IRF7, 4 μg Flag-TBK1, 4 μg HA-RPZ5 or empty vector and 1 μg HA-Ub. At 18 h posttransfection, the cells were treated with DMSO or MG132 for 6 h. Cell lysates were IP with anti-Myc affinity gels. Then, the immunoprecipitates and whole-cell lysates (WCL) were analyzed by IB with the Abs indicated. (C) RPZ5 mediates K48-linked ubiquitination of IRF7 in vivo. HEK 293T cells were transfected with 4 μg Myc-IRF7, 4 μg Flag-TBK1, 4 μg HA-RPZ5 or empty vector and 1 μg HA-Ub, HA-Ub-K48O, or HA-Ub-K63O. At 18 h posttransfection, the cells were treated with MG132 for 6 h. At 24 h posttransfection, cell lysates were immunoprecipitated with anti-Myc-affinity gel. Then, the immunoprecipitates and WCL were analyzed by IB with the Abs indicated. All experiments were repeated at least three times with similar results.
Overexpression of RPZ5 inhibits host antiviral response against SVCV infection in EPC cells.Since RPZ5 is involved in the K48-linked ubiquitination of IRF7 and negatively regulates RLR-induced IFN activation, we determined the role of RPZ5 in the cellular antiviral response. A previous study showed that SVCV efficiently infects EPC cells (29). Using cytopathic effect (CPE) assays, we found that the overexpression of RPZ5 increased the CPE compared to the empty vector after EPC cells were infected with SVCV (Fig. 7A). This was confirmed by the titer of SVCV, which increased (30-fold) in the supernatant of RPZ5-overexpressed cells compared with that in the control cells (Fig. 7B). In addition, we analyzed the expression of ifn and vig1, marker genes induced by viral infection (30, 31). As shown in Fig. 7C and D, overexpression of RPZ5 in EPC cells reduced the transcription levels of ifn and vig1 induced by SVCV infection. These results suggest that RPZ5 plays an important role in the cellular antiviral response by attenuating the expression of key antiviral genes and facilitating the replication of SVCV.
RPZ5 negatively regulates the cellular antiviral response. (A and B) Virus replication was enhanced by overexpression of RPZ5. EPC cells seeded in 24-well plates overnight were transfected with 0.5 μg of pcDNA3.1-RPZ5 or empty vector. At 24 h posttransfection, cells were infected with SVCV (MOI, 0.001) for 48 h. (A) Then, cells were fixed with 4% PFA and stained with 1% crystal violet. (B) Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured by a plaque assay. The experiments were performed for three times with similar results. (C and D) Overexpression of RPZ5 suppresses the expression (Exp) of ifn (C) and vig1 (D) induced by SVCV infection in EPC cells. EPC cells seeded in 6-well plates overnight were transfected with 2 μg of pcDNA3.1-RPZ5 or empty vector and infected with SVCV (MOI, 1) at 24 h posttransfection. At 24 h after infection, total RNA was extracted to examine the mRNA levels of cellular ifn and vig1. The relative (Rel.) transcriptional levels were normalized to the transcriptional level of the β-actin gene and were represented as fold induction relative to the transcriptional level in the control cells, which was set to 1. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from the control values (*, P < 0.05). Experiments were repeated at least three times with similar results.
DISCUSSION
In this study, we determined that zebrafish RPZ5 was induced upon SVCV stimulation and that it negatively regulates the RLR pathway. Furthermore, RPZ5 can interact with IRF7 and promote its K48-dependent ubiquitination and subsequent proteasome-dependent degradation. RPZ5 is involved in the IFN negative-feedback system that abrogates excessive IFN production because it targets the central transcription regulator of IRF7.
Fish rpz is a founding member of a novel gene family. To date, rpz homologs have only been identified in teleosts, and the zebrafish rpz family has five members (24). A missense mutation in the rpz gene of zebrafish could cause overgrowth of both the axial and the fin ray skeleton of fish (24). Interestingly, the function of rpz is unknown. In the current study, we found that zebrafish RPZ5 negatively regulates the cellular antiviral immune response. These findings may provide evidence that rpz is a multifunctional gene family.
Although the innate immune response is indispensable to host defense against pathogen invasion, IFN production must be tightly regulated because excessive IFN expression can be harmful or even fatal to the host. IRF7 is an important transcription factor that regulates IFN expression in response to viral infection (17). Thus, the regulation of IRF7 is one of the critical mechanisms that maintains IFN production at the appropriate level. FOXO3 negatively regulates the stability of IRF7 mRNA, thus inhibiting the robust production of IFN during viral infection (32). IRF7 also undergoes various posttranslational modifications, such as phosphorylation, ubiquitination, sumoylation, and acetylation (16). Fish IRF7 is a true ortholog of mammalian IRF7. A recent study showed that fish IRF7 is also involved in regulating IFN expression and undergoes phosphorylation (33). Nevertheless, little is known about the regulation mechanisms of IRF7 activity in fish. In the current study, we identified a novel negative-regulatory mechanism of the cellular antiviral response by zebrafish RPZ5 via targeting IRF7.
Indeed, protein ubiquitination has emerged as a key mechanism for regulating immune responses (34). For instance, the homolog to E6-associated protein C terminus (HECT) domain ubiquitin E3 ligase, RAUL, catalyzes the K48-linked polyubiquitination and degradation of both IRF7 and IRF3 (35). Ubiquitination requires the sequential actions of three enzymes, Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), and Ub ligase (E3), and E3 dictates which target protein are ubiquitinated. There are two types of E3 ligases, the HECT domain and really interesting new gene (RING) finger domain E3 Ub ligases (36). A BLASTP search revealed that zebrafish RPZ5 has no classical domain for the existing E3 ligase. Hence, whether RPZ5 acts as a new unknown E3 ligase or recruits another E3 ligase to catalyze the ubiquitination of IRF7 deserves further research.
Like other transcription factors, IRF7 undergoes various posttranslational modifications, the most important of which is phosphorylation (16). Several kinases, including TBK1, IKKi, and IRAK1, are involved in the phosphorylation of IRF7 (37, 38). The phosphorylation unmasks a binding domain of IRF7 and leads to the formation of a homodimer or heterodimer with IRF3, subsequently inducing the expression of IFN and cleaning infected viruses (39). Correspondingly, host cells have evolved diverse mechanisms to extinguish the IFN response by targeting distinct components in the RLR-induced antiviral signaling pathways. Our results showed that RPZ5 has no effect on the protein level of IRF7 but promotes its ubiquitination and subsequent proteasome-dependent degradation only in the presence of TBK1. We speculate that RPZ5 degradation of the TBK1-mediated phosphorylation of IRF7 is a host cellular mechanism to balance IFN production. However, viruses likely take advantage of the host negative-regulatory proteins for better viral replication. Our study showed that overexpression of RPZ5 facilitates the replication of SVCV. Therefore, RPZ5 inhibiting IFN expression may be an evasion mechanism mediated by viral proteins against the IFN system, but this needs to be further investigated.
In summary, this study demonstrates that zebrafish RPZ5 targets IRF7 and triggers the ubiquitination and degradation of IRF7. The overall function of this process is to turn off or limit the excess production of IFN. Thus, we report a novel role for zebrafish RPZ5 in regulating cellular antiviral responses.
MATERIALS AND METHODS
Cells, viruses, and zebrafish.Human embryonic kidney (HEK) 293T cells were provided by Xing Liu (Institute of Hydrobiology, Chinese Academy of Sciences) and were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Zebrafish liver (ZFL) cells (American Type Culture Collection [ATCC]) were cultured at 28°C in 5% CO2 in Ham’s F-12 nutrient mixture medium (Invitrogen) supplemented with 10% FBS. Epithelioma papulosum cyprini (EPC) cells were obtained from the China Center for Type Culture Collection (CCTCC) and were maintained at 28°C in 5% CO2 in medium 199 (Invitrogen) supplemented with 10% FBS. Spring viremia of carp virus (SVCV) was propagated in EPC cells until cytopathic effect (CPE) was observed; then, the culture medium with cells was harvested and stored at –80°C until needed.
Plasmid construction and reagents.The open reading frame (ORF) of zebrafish RPZ5 (GenBank accession number NM_001110124.1) was generated by PCR and then cloned into the pcDNA3.1(+) (Invitrogen), pCMV-Myc (Clontech), or pCMV-HA (Clontech) vector, respectively. The ORFs of zebrafish MAVS (GenBank accession number NM_001080584.2), TBK1 (GenBank accession number NM_001044748.2), MITA (GenBank accession number NM_001278837.1), IRF3 (GenBank accession number NM_001143904), and IRF7 (GenBank accession number NM_200677.2) were also subcloned into the pcDNA3.1(+), pCMV-HA, pCMV-Tag2C (Clontech), or pDsRed-N1 (Clontech) vector, respectively. For subcellular localization, the ORF of RPZ5 was inserted into pEGFP-N3 vector (Clontech). The pDsRed-ER and pDsRed-Golgi plasmids were purchased from BD Clontech. The pCMV-HA vector containing the truncated mutants of IRF7 and the plasmids containing IFNφ1pro-Luc and IFN-stimulated response element (ISRE)-Luc in the pGL3-Basic luciferase reporter vector (Promega) were constructed as described previously (19, 22). The Renilla luciferase internal control vector (pRL-TK) was purchased from Promega. HA-Ub-K48O and HA-Ub-K63O were expression plasmids for HA-tagged Lys-48- and Lys-63-only ubiquitin mutants (all lysine residues except Lys-48 or Lys-63 are mutated). All constructs were confirmed by DNA sequencing. MG132 and poly(I·C) were purchased from Sigma-Aldrich and used at final concentrations of 20 μM/ml and 1 μg/ml, respectively.
Luciferase activity assay.EPC cells were seeded in 24-well plates overnight and cotransfected with various plasmids at a ratio of 10:10:1 (expression vectors of MAVS/TBK1/MITA/IRF3/IRF7:IFNφ1pro/ISRE-Luc:pRL-TK). The empty vector pcDNA3.1(+) was used to ensure that there were equivalent amounts of total DNA in each well. Transfection of poly(I·C) was performed at 24 h before cell harvest. At 48 h posttransfection, the cells were washed with phosphate-buffered saline (PBS) and lysed for measuring luciferase activity by the Dual-luciferase reporter assay system (Promega), according to the manufacturer’s instructions. Firefly luciferase activity was normalized on the basis of Renilla luciferase activity. At least three independent experiments were performed, and the data from one representative experiment were used for statistical analysis.
RNA extraction, reverse transcription, and qPCR.Total RNA was extracted by the TRIzol reagent (Invitrogen). First-strand cDNA was synthesized by using a GoScript reverse transcription system (Promega), according to the manufacturer’s instructions. qPCR was performed with Fast SYBR green PCR master mix (Bio-Rad) on the CFX96 real-time system (Bio-Rad). The PCR conditions were as follows: 95°C for 5 min and then 40 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 20 s. The β-actin gene was used as an internal control. The relative fold changes were calculated by comparison to the corresponding controls using the 2−ΔΔCT method. At least three independent experiments were performed, and the data from one representative experiment were conducted for statistical analysis. The following gene-specific primers were utilized for the qPCR: DrRPZ5, 5′-CATAACCCAATACGAGAACAC-3′ and 5′-CTTCCTGCTCCTTTCGTCC-3′; DrIFN1, 5′-GAATGGCTTGGCCGATACAGGATA-3′ and 5′-TCCTCCACCTTTGACTTGTCCATC-3′; DrIRF7, 5′-CCTTGGTGACGCGGATGT-3′ and 5′-GGAAGTTTGTCTTCCATTTTGCTTT-3′; Drβ-actin, 5′-CACTGTGCCCATCTACGAG-3′ and 5′-CCATCTCCTGCTCGAAGTC-3′; RPZ5-EPC, 5′-GGAATTATAGCAGTCATGG-3′ and 5′-GATGCTCCACGTCCGTC-3′; IFN-EPC, 5′-ATGAAAACTCAAATGTGGACGTA-3′ and 5′-GATAGTTTCCACCCTTTCCTTAA-3′; VIG1-EPC, 5′-AGCGAGGCTTACGACTTCTG-3′ and 5′-GCACCAACTCTCCCAGAAAA-3′; and β-actin-EPC, 5′-CTCCATTGAACTGCTGAAAGATG-3′ and 5′-CAAATAACTGTCTTCATTTCGCTCAT-3′.
Transient transfection and virus infection.Transient transfections were performed in EPC cells seeded in 6-well or 24-well plates or in ZFL cells seeded in 6-well plates by using X-tremeGENE high-performance (HP) DNA transfection reagent (Roche), according to the manufacturer’s protocol. For the antiviral assay using 24-well plates, EPC cells were transfected with 0.5 μg pcDNA3.1-RPZ5 or the empty vector. At 24 h posttransfection, cells were infected with SVCV at a multiplicity of infection (MOI) of 0.001. After 2 or 3 days, supernatant aliquots were harvested for detection of virus titers, and the cell monolayers were fixed by 4% paraformaldehyde (PFA) and stained with 1% crystal violet for visualizing CPE. For virus titration, 200 μl of culture medium was collected at 48 h postinfection and used for the plaque assay. The supernatants were subjected to 3-fold serial dilutions and then added (100 μl) onto a monolayer of EPC cells cultured in a 96-well plate. After 48 or 72 h, the medium was removed, and the cells were washed with PBS, fixed by 4% PFA, and stained with 1% crystal violet. The virus titer was expressed as the 50% tissue culture infective dose (TCID50/ml). The results are representative of three independent experiments.
RNA interference experiments.EPC cells were seeded in 6-well plates overnight and transfected with 100 nM siRNA of RPZ5 or the negative control (si-Nc) by using X-tremeGENE HP DNA transfection reagent (Roche), according to the manufacturer’s protocol. siRNA of RPZ5 and si-Nc were obtained from RiboBio Co., Ltd. (Guangzhou, China). The following sequences were targeted for EPC RPZ5: siRPZ5#1, AGCAGAACTTGACCGTGA; and siRPZ5#2, GAAGTTCATAAGCCAGTAT.
Coimmunoprecipitation assay.The HEK 293T cells seeded in 10-cm2 dishes overnight were transfected with a total of 10 μg of the plasmids indicated in Fig. 4A and Fig. 6B and C. At 24 h posttransfection, the medium was removed carefully, and the cell monolayer was washed twice with 10 ml ice-cold PBS. Then, the cells were lysed in 1 ml of radioimmunoprecipitation (RIPA) lysis buffer (1% NP-40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate [Na3VO4], 1 mM phenylmethylsulfonyl fluoride [PMSF], 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Sigma-Aldrich) at 4°C for 1 h on a rocker platform. The cellular debris was removed by centrifugation at 12,000 × g for 15 min at 4°C. The supernatant was transferred to a fresh tube and incubated with 30 μl anti-Flag-agarose beads or anti-Myc affinity gel (Sigma-Aldrich) overnight at 4°C with constant agitation. These samples were further analyzed by immunoblotting (IB). Immunoprecipitated proteins were collected by centrifugation at 5,000 × g for 1 min at 4°C, washed three times with lysis buffer, and resuspended in 50 μl of 2× SDS sample buffer. The immunoprecipitates and whole-cell lysates were analyzed by IB with the indicated antibodies (Abs).
Immunoblot analysis.Immunoprecipitates or whole-cell lysates were separated by 10% SDS-PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranes were blocked for 1 h at room temperature in TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20 [pH 7.5]) containing 5% nonfat dry milk, probed with the indicated primary Abs at an appropriate dilution overnight at 4°C, washed three times with TBST, and then incubated with secondary Abs for 1 h at room temperature. After three additional washes with TBST, the membranes were stained with the Immobilon Western chemiluminescent horseradish peroxidase (HRP) substrate (Millipore) and detected by using an ImageQuant LAS 4000 system (GE Healthcare). Abs were diluted as follows: anti-β-actin (Cell Signaling Technology) at 1:1,000, anti-Flag/HA (Sigma-Aldrich) at 1:3,000, anti-Myc (Santa Cruz Biotechnology) at 1:2,000, and HRP-conjugated anti-mouse IgG (Thermo Scientific) at 1:5,000. The results are representative of three independent experiments.
In vitro protein dephosphorylation assay.Transfected HEK 293T cells were lysed as described above, except that the phosphatase inhibitors (Na3VO4 and EDTA) were omitted from the lysis buffer. Protein dephosphorylation was carried out in 100-μl reaction mixtures consisting of 100 μg of cell protein and 10 units of calf intestinal alkaline phosphatase (CIP; Sigma-Aldrich). The reaction mixtures were incubated at 37°C for 40 min, followed by immunoblot analysis.
Fluorescent microscopy.EPC cells were plated onto coverslips in 6-well plates and transfected with the plasmids indicated in the figures for 24 h. Then, the cells were washed twice with PBS and fixed with 4% PFA for 1 h. After being washed three times with PBS, the cells were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) for 15 min in the dark at room temperature. Finally, the coverslips were washed and observed with a confocal microscope under a 63× oil immersion objective (SP8; Leica).
Statistics analysis.The luciferase and qPCR assay data are expressed as the mean ± standard error of the mean (SEM). Error bars indicate the SEM (n = 3, biologically independent samples). The P values were calculated by one-way analysis of variance (ANOVA) with Dunnett’s post hoc test (SPSS Statistics, version 19; IBM). A P value of <0.05 was considered statistically significant.
ACKNOWLEDGMENTS
We thank Yan-Yi Wang (Wuhan Institute of Virology, Chinese Academy of Sciences) for providing plasmids (HA-Ub, HA-Ub-K48O, and HA-Ub-K63O) and Fang Zhou (Institute of Hydrobiology, Chinese Academy of Sciences) for assistance with confocal microscopy analysis.
The National Key Research and Development Program of China provided funding to Shun Li under grant number 2018YFD0900504. The National Natural Science Foundation of China provided funding to Long-Feng Lu under grant number 31802338.
We declare no financial or commercial conflicts of interest.
FOOTNOTES
- Received 1 August 2019.
- Accepted 9 August 2019.
- Accepted manuscript posted online 14 August 2019.
- Copyright © 2019 American Society for Microbiology.
