Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production

Abstract

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.

Keywords: GCRV; MAVS; VP35; immune evasion; interferon.

Figure 1

Grass carp reovirus (GCRV) 35-kDa protein (VP35) inhibits polyinosinic-polycytidylic acid (poly I:C) or spring viremia of carp virus- (SVCV-) induced interferon (IFN) expression. (A) Quantitative real-time PCR (qPCR) detects the transcription level of s11 after grass carp ovary (GCO) infection. GCO cells were seeded on six-well plates overnight and infected with GCRV [100 μl of the filtered virus containing supernatant of frozen and thawed GCO cells, which was diluted 100 times with phosphate buffered saline (PBS)]. At 1, 3, and 7 day post-infection (dpi), total RNAs were extracted for qRCR detection. (B–G) Overexpression of VP35 inhibits poly I: C or SVCV-induced IFN1pro, DrIFNϕ1pro, and interferon-stimulated response element (ISRE) (D,G) activation. Inoculated GCO cells into a 24-well plate overnight, and transfected with 250 ng IFN1pro-Luc (B,E), DrIFNϕ1pro (C,F), or ISRE-Luc (D,G) and 25 ng pRL-TK, plus 250 ng VP35-pcDNA3.1 (+), or pcDNA3.1 (+) (control vector). After 24 h, the cells were untreated or treated with SVCV [multiplicity of infection (MOI) = 1] or poly I:C (1 μg/ml). The cells were collected and then lysed for luciferase assay. (H–L) Overexpression of VP35 inhibits the expression of ifn and other IFN-stimulated genes (ISGs) induced by poly I:C. The EPC cells seeded in six-well plates overnight were transfected with 2 μg VP35-pcDNA3.1 or empty vector and transfected with poly I:C at 24 h post-transfection. At 24 h after stimulation, total RNAs were extracted to examine the mRNA levels of cellular ifn (H), vig1 (I), rig-i (J), isg15 (K), and irf7 (L). The relative transcriptional levels were normalized to the transcription of β-actin 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 a significant difference from the control (*p < 0.05).

Figure 2

VP35 suppresses MAVS-activated IFN induction. (A–C) GCO cells were seeded into a 24-well plate and co-transfected with 250 ng expression plasmids MAVS/TRAF family member-associated NF-κB activator (TANK) binding kinase 1 (TBK1)/IFN regulatory factor 3 (IRF3) with 250 ng VP35-pcDNA3.1(+) or empty vector, plus 250 ng IFN1pro-Luc (A), DrIFNϕ1pro-Luc (B), and ISRE-Luc (C), and 25 ng pRL-TK. After 24 h, the cells were lysed for monitoring luciferase activity. (D–F) Overexpression of VP35 inhibited the activities of IFN1pro, DrIFNϕ1pro and ISRE induced by MAVS in a dose-dependent manner. Inoculated GCO cells in a 24-well plate overnight, and co-transfected with 250 ng IFN1pro-Luc (D), DrIFNϕ1pro-Luc (E), and ISRE-Luc (F), 25 ng pRL-TK, 250 ng MAVS and VP35-pcDNA3.1 (+) (0, 100, 200, or 400 ng). Data are expressed as mean ± SEM, n = 3. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*p < 0.05).

Figure 3

VP35 is distributed throughout the cells and interacts with (RIG-I)-like receptor (RLR) molecules. (A) Inoculated human embryonic kidney (HEK) 293T cells into 10-cm² dishes and transfected with the indicated plasmids (5 μg each). After 24 h, the cells were lysed and immunoprecipitated (IP) with Anti-Flag Affinity Gel. Then the immunoprecipitate and whole-cell lysates (WCLs) were analyzed by immunoblotting (IB) with anti-Flag and anti-Myc antibodies (Abs), respectively. (B) EPC cells were seeded into a six-well plate, and transfected with 1 μg MAVS-DsRed, TBK1-DsRed, and IRF3-DsRed and 1 μg VP35-EGFP or empty vector. After 24 h, the cells were fixed and analyzed by confocal microscopy. The green signal indicates the over-expressed VP35 protein, the red signal indicates the over-expressed MAVS, TBK1, and IRF3, and the blue indicates the nuclear area (original magnification 63 ×; oil immersion objective). Scale bar, 10 μm. (C) Schematic representation of full-length MAVS and its mutants. (D) The 221–400 aa and 401–585 aa of MAVS are responsible for its interaction with VP35. The experiments were performed similarly as described above for (A). All experiments were repeated at least three times with similar results.

Figure 4

VP35 degrades MAVS through the autophagy pathway. (A) EPC cells were seeded in six-well plates overnight and co-transfected with 1 μg of MAVS/TBK1/IRF3-HA and 1 μg of empty vector or VP35-Myc for 24 h. The WCLs were subjected to IB with the anti-HA, anti-Myc, and anti-β-actin Abs. (B,C) EPC cells were seeded into six-well plates overnight and co-transfected with 1 μg MAVS-HA (B) or TBK1-HA (C) plus various concentration of VP35-Myc (0, 1, or 2 μg, empty vector was used to make up the rest). After 24 h, the WCLs were subjected to IB with anti-Myc, anti-HA, and anti-β-actin Abs. (D) Effects of inhibitors on VP35-mediated degradation of MAVS. EPC cells were seeded in six-well plates overnight and co-transfected with 1 μg MAVS-HA and 1 μg VP35-Myc. At 18 h post-transfection, the cells were treated with the dimethyl sulfoxide (DMSO), MG132 (20 μM), 3-MA (2 mM), or NH₄Cl (20 mM) for 6 h prior to being harvested for IB analysis of WCLs with the anti-HA, anti-Myc, and anti-β-actin Abs. (E) VP35-induced MAVS degradation is rescued by 3-MA in a dose-dependent manner. EPC cells were seeded in six-well plates overnight and co-transfected the indicated plasmids. At 18 h post-transfection, the cells were treated with DMSO or 3-MA (1, 2, or 4 mM) for 6 h. Then, the cells were harvested for IB with the Abs indicated. All experiments were repeated at least three times with similar results.

Figure 5

VP35 affects MAVS-mediated signal transmission and the host IFN production. (A) HEK 293T was seeded in 10-cm² dishes and transfected with 4 μg RIG-I-Flag, 4 μg MAVS-Myc, and 4 μg VP35-HA. After 24 h, the cells were lysed for co-immunoprecipitation (Co-IP) with Anti-Flag Affinity Gel. Then the immunoprecipitate and WCLs were analyzed by IB with anti-Flag, anti-HA, anti-Myc, and anti-β-actin Abs. (B–F) EPC cells were seeded in six-well plates and transfected with 2 μg MAVS-Myc and VP35-HA, after 24 h, total RNAs were extracted to examine the transcriptional levels of ifn/vig1/rig-i/isg15/irf7. The relative 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 control values (*p < 0.05).

Figure 6

VP35 blocks MAVS-induced cellular antiviral immune response. (A) EPC cells seeded in 24-well plates overnight were transfected with 0.5 μg of MAVS-HA and 0.5 μg VP35-pcDNA3.1(+) or pcDNA3.1(+). At 24 h post-transfection, cells were infected with SVCV (MOI = 0.001) for 48 h. Then, cells were fixed with 4% paraformaldehyde (PFA) and stained with 1% crystal violet. (B) Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured according to the method of Reed and Muench. (C) EPC was seeded in six-well plates and transfected with 2 μg of MAVS-HA and 2 μg of VP35-Myc or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 1). After 24-h infection, the WCLs were detected by IB with the anti-N, anti-P, anti-Myc, anti-HA, and anti-β-actin Abs, respectively. (D–H) The same samples were prepared similarly as described above for (C). Total RNAs were extracted to examine the mRNA levels of cellular n, p, m, g, and l. The relative 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 control values (*p < 0.05).

Figure 7

VP35 reduces host IFN response and promotes virus proliferation. (A,B) EPC cells seeded in 24-well plates overnight were transfected with 0.5 μg VP35-pcDNA3.1 (+) or pcDNA3.1 (+) vector. At 24 h post-transfection, 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 according to the method of Reed and Muench. (C–G) EPC cells seeded into six-well plates overnight were infected with SVCV (MOI = 1). After 24 h, total RNAs were extracted to examine the transcriptional levels of cellular ifn (C), vig1 (D), rig-i (E), isg15 (F), irf7 (G) by qPCR. The relative transcription levels were normalized to the transcription level of the β-actin gene and are represented as fold induction relative to the transcription level in control cells, which was set to 1. Data are expressed as mean ± SEM, n = 3. Asterisks indicate significant differences from control (*p < 0.05).