Seneca Valley Virus Induces DHX30 Cleavage to Antagonize Its Antiviral Effects

Abstract

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3C^pro-interacting protein. 3C^pro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3C^pro-interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3C^pro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Keywords: 3Cpro; DHX30; LC-MS/MS; RIP-seq; RNA binding; Seneca Valley virus; antiviral; cleavage.

FIG 1

Identification of host proteins interacting with SVV 3C^pro. (A) Silver stain of the eluted fraction following HA affinity purification of the indicated bait proteins. The expression of 3C-DM was confirmed by Western blotting. (B and C) HEK293T cells were cotransfected with empty plasmids, HA-3C, or HA-3C-DM and Flag-DHX30 or Flag-DDX21 for 24 h, and then the cells were collected for Co-IP analysis. (D) HEK293T cells were cotransfected with HA-3C or HA-3C-DM and Flag-DHX30 for 24 h, and then the cells were analyzed by confocal microscope after staining with anti-HA antibodies and anti-Flag antibodies. Scale bar = 10 μm.

FIG 2

SVV 3C^pro cleaved DHX30 at a specific site through its protease activity. (A) HEK293T cells were cotransfected with Flag-DHX30 and increased amounts of HA-3C for 24 h, and then the cells were collected for Western blotting. (B) HEK293T cells were cotransfected with Flag-DHX30 and empty plasmids, HA-3C, or 3C mutants for 24 h, and then the cells were collected for Western blotting. (C) HEK293T cells were cotransfected with Flag-DHX30 and empty plasmids or HA-3C for 16 h, and then the cells were incubated with Z-VAD-FMK for 8 h. The cells were then collected for Western blotting. (D) HEK293T cells were transfected with Flag-DHX30 for 24 h. The cell lysates were incubated with purified 3C^pro at 37°C for 3 h and then prepared for Western blotting using the indicated antibodies. (E) HEK293T cells were transfected with Flag-DHX30 for 24 h, and then the cells were infected with 1 MOI SVV for the indicated times. The cells were collected for Western blotting. (F to H) HEK293T cells, SK6 cells, or BHK21 cells were infected with SVV (MOI = 1) for the indicated times, and then the cell lysates were prepared for Western blotting. (I) HEK293T cells were infected with SVV (MOI = 1) for the indicated times. The mRNA level of DHX30 was measured by real-time PCR analysis. (J) The schematic representation of DHX30 and its mutants. (K) HEK293T cells were cotransfected with HA-3C and plasmids encoding DHX30 mutants for 24 h, and then the cells were collected for Western blotting. (L) HEK293T cells were transfected with Flag-DHX30-Q220A for 24 h, and then the cell lysates were prepared for Western blotting.

FIG 3

DHX30 inhibited SVV replication. (A and B) HEK293T cells were transfected with Flag-DHX30 for 24 h, and then the cells were infected with SVV (MOI = 0.1) for the indicated times. The cells were collected for Western blotting, and the viral titers were determined by plaque assay. (C and D) HEK293T cells were transduced with shNC lentivirus or shDHX30 lentivirus for 48 h, and then the cells were infected with SVV (MOI = 0.1) for the indicated times. The cells were collected for Western blotting, and the virus titers were determined by plaque assay. (E) HEK293T cells were transfected with empty plasmids or Flag-DHX30 for 24 h, and then the cells were infected with SVV-GFP (MOI = 0.1) for 6 h. Then fluorescence microscopy images were collected. Scale bar = 50 μm. (F and G) SK6 cells were performed the same as (A and B). Data are shown as means ± SD. *, P < 0.05; **, P < 0.01.

FIG 4

3C-mediated DHX30 cleavage products lost their ability to inhibit SVV propagation. (A to D) HEK293T cells were transfected with plasmids encoding DHX30 mutants for 24 h, and then the cells were infected with SVV (MOI = 0.1) for the indicated times. The cells were collected for Western blotting, and the viral titers were determined by plaque assay. Data are shown as means ± SD. *, P < 0.05; **, P < 0.01.

FIG 5

DHX30 helicase activity was essential for its anti-SVV effects. (A) The schematic representation of DHX30 mutant. (B and C) HEK293T cells were transfected with plasmids encoding DHX30 mutants for 24 h, and then the cells were infected with SVV (MOI = 0.1) for the indicated times. The cells were collected for Western blotting, and the virus titers were determined by plaque assay. (D) HEK293T cells were transfected with empty plasmids, Flag-DHX30, or Flag-DHX30-DAVH for 24 h, and then the cells were infected with SVV-GFP (MOI = 0.1) for 6 h. The fluorescence microscopy images were then collected. Scale bar = 50 μm. Data are shown as means ± SD. **, P < 0.01.

FIG 6

DHX30 inhibited SVV replication at the early stage of the life cycle. (A to D) HEK293T cells were transfected with empty plasmids or Flag-DHX30 for 24 h. (A) The cells were infected with SVV at 10 MOI and adsorbed to cells for 1 h at 4°C. After adsorption, the cells were washed five times with cold PBS. For entry assays, the washed cells were incubated with a prewarmed medium containing 5% FBS at 37°C for 1 h. The cells were then immediately chilled on ice and washed with cold PBS three times followed by three washes each of cold alkaline high-salt solution. The cells were collected for RT-qPCR assay. (B and C) The cells were infected with SVV (MOI = 1) for the indicated times, The cells were collected for RT-qPCR assay and Western blotting. (D) The cells were infected with SVV at 4°C to synchronize the infection, washed, and treated with cycloheximide (CHX). Samples were collected at the indicated time points for RT-qPCR. (E) Bicistronic plasmid construction. (F) HEK293T cells were cotransfected with empty plasmids or Flag-DHX30 and pIRIGF or SVV-5’UTR-Luc for 36 h, and then the cells were collected for dual-luciferase activity analysis. Data are shown as means ± SD. *, P < 0.05; ***, P < 0.001.

FIG 7

DHX30 bound SVV dsRNA and inhibited viral genome replication. (A) HEK293T cells were transfected with empty plasmids or Flag-DHX30 for 24 h, and then the cells were infected with SVV (MOI = 1) for 8 h. The cell lysates were incubated with anti-Flag antibodies. The DHX30-binding RNA pulled down by antibodies was amplified by RT-PCR with SVV 5’UTR specific primers. Exogenous DHX30 expression was determined by Western blotting. (B and C) Same as (A). (D) Map of DHX30 binding sites in SVV genome by RIP-seq of RNA isolated from SVV-infected DHX30 overexpressing HEK293T cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (E) HEK293T cells were transfected with pcDNA3.0-5’UTR or Flag-DHX30 for 24 h. Then the cell lysates were incubated with anti-Flag antibodies. The DHX30-binding RNA pulled down by antibodies was amplified by RT-PCR with SVV 5’UTR specific primers. Exogenous DHX30 expression was determined by Western blotting. (F) HEK293T cells were transfected with empty plasmids or Flag-DHX30 for 24 h, and then the cells were infected with SVV (MOI = 5) for 6 h. After staining with anti-dsRNA antibodies and anti-Flag antibodies, the cells were analyzed by a confocal microscope. Scale bar = 10 μm. (G) HEK293T cells were cotransfected with empty plasmids or Flag-DHX30 and HA-3D for 24 h. Subsequently, the interaction between DHX30 and 3D was measured by anti-Flag immunoprecipitation. (H and I) HEK293T cells were cotransfected with empty plasmids or Flag-DHX30-Q220A and HA-3D for 24 h, and then the cells were infected with SVV (MOI = 10) for 10 h. Subsequently, the interaction between DHX30 and 3D was measured by anti-Flag immunoprecipitation.