Zebrafish RPZ5 Degrades Phosphorylated IRF7 To Repress Interferon Production

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.

Keywords: IFN; IRF7; RPZ5; interferons; negative regulator; ubiquitination; zebrafish.

FIG 1

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.

FIG 2

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.

FIG 3

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.

FIG 4

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.

FIG 5

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.

FIG 6

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.

FIG 7

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.