SVCV infection causes hypoxia-induced symptoms and promotes hypoxia-inducible gene expression in zebrafish. (A-C) SVCV infection caused symptoms in zebrafish, similar to those observed under hypoxic conditions. After 24 h of starvation, adult zebrafish (3 mpf) were injected intraperitoneally with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). Twenty-four hours later, infected zebrafish were observed swimming at the surface of the water with their opercula erect and breathing rapidly. Based on the video recordings, we counted the number of times the gill covers opened and closed in three groups of zebrafish. (D) qPCR analysis of SVCV-P, SVCV-G, SVCV-N, SVCV-M, and SVCV-L mRNA in the brain of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (E) qPCR analysis of ifn1 and lta mRNA in the brain of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (F) qPCR analysis of vegfaa and phd3 mRNA in the brain of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (G) qPCR analysis of SVCV-P, SVCV-G, SVCV-N, SVCV-M, and SVCV-L mRNA in the spleen of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (H) qPCR analysis of ifn1 and lta mRNA in the spleen of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (I) qPCR analysis of vegfaa and phd3 mRNA in the spleen of zebrafish (3 mpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL, 10 µL/individual). (J) Representative images of Tg (HRE-sv40mp : GFP) zebrafish larvae (3 dpf) infected with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (K) Quantitation of total intensity in (J).

SVCV infection enhances hypoxia signaling in zebrafish and zebrafish cells. (A) qPCR analysis of SVCV-N, SVCV-P, SVCV-M, SVCV-G, and SVCV-L mRNA in zebrafish larvae (3 dpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (B) qPCR analysis of ldha, glut1, vegfaa, phd3, and pdk1 mRNA in zebrafish larvae (3 dpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (C) Heatmap for the selected responsive genes of RIG-I-like receptor signaling pathway in zebrafish larvae (3 dpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (D) Heatmap for the selected responsive genes of HIF-mediated hypoxia signaling pathway in zebrafish larvae (3 dpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (E) Gene Ontology (GO) enrichment analyses for the DEGs were performed using the cluster Profiler version 3.8. in zebrafish larvae (3 dpf) with or without infection with SVCV (5 × 10⁷ TCID₅₀/mL). (F) qPCR analysis of SVCV-G mRNA in ZFL cells infected with an increasing amount of SVCV. (G) qPCR analysis of ifn1 mRNA in ZFL cells infected with an increasing amount of SVCV. (H, I) qPCR analysis of vegfaa (H) and glut1 (I) mRNA in ZFL cells infected with an increasing amount of SVCV. (J) qPCR analysis of SVCV-G mRNA in ZF4 cells infected with an increasing amount of SVCV. (K) qPCR analysis of ifn1 mRNA in ZF4 cells infected with an increasing amount of SVCV. (L, M) qPCR analysis of vegfaa (L) and glut1 (M) mRNA in ZF4 cells infected with an increasing amount of SVCV. (N) HRE reporter activity in ZF4 cells with or without infection of SVCV.

SVCV infection enhances glucose uptake, glycolytic rate, and intracellular and mitochondrial ROS levels in ZF4 cells. (A) Glucose uptake in ZF4 cells with or without SVCV infection as detected by uptake of the fluorescent glucose analog 2-NBDG using fluorescence microscopy (n = 3). Scale bar = 100 µm. (B, C) Proton efflux rate (PER) changes in ZF4 cells (n = 3) with or without SVCV infection as measured using Seahorse XFe24 Extracellular Flux Analyzer (B). Statistical analysis of basal glycolysis and compensatory glycolysis were shown in (C). (D, E) The levels of intracellular ROS in ZF4 cells infected with SVCV with increasing doses were detected by flow cytometry analysis. Quantitation of intracellular ROS levels in (D) was shown in (E). (F, G) The levels of mitochondrial ROS in ZF4 cells infected with SVCV with increasing doses were detected by flow cytometry analysis. Quantitation of mitochondrial ROS levels in (F) was shown in (G).

SVCV-G protein stabilizes hif1αa and hif1αb. (A, B) Immunoblotting (IB) of Myc-hif1αa (A) and Myc-hif1αb (B) in HEK293T cells transfected with the plasmid expressing Myc-hif1αa or Myc-hif1αb together with increasing amounts of Flag-SVCV-G (Flag empty vector [-] was used as a control). (C, D) IB of Myc-hif1αa (C) and Myc-hif1αb (D) in HEK293T cells transfected with the plasmid expressing Myc-hif1αa or Myc-hif1αb together with increasing amounts of Flag-SVCV-P (Flag empty vector [-] was used as a control). (E, F) IB of Myc-hif1αa (E) and Myc-hif1αb (F) in HEK293T cells transfected with the plasmid expressing Myc-hif1αa or Myc-hif1αb together with increasing amounts of Flag-SVCV-N (Flag empty vector [-] was used as a control). (G, H) HRE reporter activity in HEK293T cells transfected with the plasmid expressing Myc-hif1αa (G) or Myc-hif1αb (H) together with Flag-SVCV-G (Flag empty vector was used as a control). (I, J) IB of the indicated proteins in HEK293T cells transfected with Myc-hif1αa together with Flag-SVCV-G for 24 h (Flag empty vector [-] was used as a control) and then treated with cycloheximide (CHX, 50 µg/mL) for an increasing time (0–6 h) (I). The relative intensities of Myc-hif1αa were determined by normalizing the intensities of Myc-hif1αa to the intensities of GAPDH (J). (K, L) IB of the indicated proteins in 293T cells transfected with Myc-hif1αb together with Flag-SVCV-G for 24 h (Flag empty vector [-] was used as a control) and then treated with CHX (50 µg/mL) for an increasing time (0–6 h) (K). The relative intensities of Myc-hif1αb were determined by normalizing the intensities of Myc-hif1αb to the intensities of GAPDH (L).

SVCV-G protein interacts with hif1αa and hif1αb to attenuate K48-linked polyubiquitination of hif1αa and hif1αb. (A) Myc-hif1αa co-localized with Flag-SVCV-G as revealed by co-localization assays. Scale bar = 10 µm. (B) Myc-hif1αb co-localized with Flag-SVCV-G as revealed by co-localization assays. Scale bar = 10 µm. (C) Myc-hif1αa interacted with Flag-SVCV-G as revealed by co-immunoprecipitation assays. (D) Myc-hif1αb interacted with Flag-SVCV-G as revealed by co-immunoprecipitation assays. (E) Endogenous interaction between hif1α and SVCV in ZFL cells. Anti-SVCV-G antibody was used for immunoprecipitation (IP), and the interaction was detected by IB using anti-hif1α antibody. (F, G) Ubiquitination analysis of hif1αa in HEK293T cells transfected with Myc-hif1αa, Flag-SVCV-G (Flag empty vector [-] was used as a control), and His-Ub (F) or His-Ub-K48 (G) for 24 h, and then treated with MG132 (20 µM) for 8 h. (H, I) Ubiquitination analysis of hif1αb in HEK293T cells transfected with Myc-hif1αb, Flag-SVCV-G (Flag empty vector [-] was used as a control), and His-Ub (H) or His-Ub-K48(I) for 24 h, and then treated with MG132 (20 µM) for 8 h.

HIF-1α inhibitor PX478 enhances the antiviral ability against SVCV infection and inhibits SVCV replication in ZFL cells. (A) qPCR analysis of phd3 mRNA in ZFL cells treated with PX478 (10 µM) for 12 h, followed by hypoxia treatment for 24 h. (B) qPCR analysis of phd3 mRNA in ZFL cells treated with PX478 (10 µM) for 12 h, followed by infected with or without SVCV for 24 h. (C-F) qPCR analysis of ifn1 (C), mxc (D), mxb (E), and lta (F) mRNA in ZFL cells treated with PX478 (10 µM) for 12 h, followed by infected with or without SVCV for 24 h. (G) qPCR analysis of SVCV-G mRNA in ZFL cells treated with PX478 (10 µM) for 12 h, followed by infected with or without SVCV for 24 h. (H) ZFL cells were treated with or without PX478 (10 µM) for 12 h, followed by those infected with SVCV for 24 h. Culture supernatant was collected, and viral titers were measured by a 50% tissue culture–infective dose (TCID₅₀) assay on EPC cells. (I) ZFL cells seeded on 35 mm glass bottom cell culture dishes were treated with PX478 (10 µM) for 12 h, followed by infected with increased gradient SVCV for 24 h. The cells were fixed and stained with anti-SVCV-G antibody and subsequent confocal microscopy analysis. Scale bar = 25 µm.

HIF-1α inhibitor PX478 enhances the antiviral ability against SVCV infection and inhibits SVCV replication in zebrafish. (A) Representative images of zebrafish larvae (3 dpf) treated with DMSO (vehicle control) or PX478 (10 µM) for 12 h, followed by infected with SVCV (5 × 10⁷ TCID₅₀/mL) for 36 h. (B) Survival (Kaplan-Meier curve) of zebrafish larvae (3 dpf) (n = 30 per group) treated with PX478 (10 µM) for 12 h, followed by infected with or without SVCV (5 × 10⁷ TCID₅₀/mL) for 52 h. (C, D) qPCR analysis of phd3 (C) and vegfaa (D) mRNA in zebrafish larvae (3 dpf) treated with DMSO (vehicle control) or PX478 (10 µM) for 12 h, followed by infected with or without SVCV (5 × 10⁷ TCID₅₀/mL) for 24 h. (E-I) qPCR analysis of ifn1 (E), ifn2 (F), mxb (G), mxc (H), and lta (I) mRNA in zebrafish larvae (3 dpf) treated with DMSO (vehicle control) or PX478(10 µM) for 12 h, followed by infected with or without SVCV (5 × 10⁷ TCID₅₀/mL) for 24 h. (J) qPCR analysis of SVCV-G mRNA in zebrafish larvae (3 dpf) treated with DMSO (vehicle control) or PX478(10 µM) for 12 h, followed by infected with or without SVCV (5 × 10⁷ TCID₅₀/mL) for 24 h.