Viperin suppresses goose astrovirus type 2 replication through inhibition of viral RdRP activity

Figures

• 

  Figure 1. GAstV-2 activates innate immune responses in vitro and in vivo. A, B Transcript levels of innate immune-related genes (IFITM5, OASL, MX, TRIM25, and Viperin) in GAstV-2-infected and control LMH cells at 24 and 48 hpi. C, D Transcript levels of innate immune-related genes in GAstV-2-infected and control GRTE cells at 24 and 48 hpi. E, F Transcript levels of innate immune-related genes in the kidney (E) and liver (F) from GAstV-2-infected and control goslings. G, H Protein levels of Viperin in GAstV-2-infected and control LMH cells at 24 and 48 hpi. I, J Protein levels of Viperin in the kidney (I) and liver (J) from GAstV-2-infected and control goslings. In panels A–D, G, and H, data are presented as mean ± SD from three biological replicates; in E, F, I, and J, data are presented as mean ± SD from five gosling samples. Student’s t-test was used for comparison between control and GAstV-2 groups (*P < 0.05, **P < 0.01).

• 

  Figure 2. GAstV-2 ORF2 plays a vital role in activating Viperin expression. A LMH cells were transfected with plasmids expressing ORF1a, ORF1b, or ORF2 for 48 h. Total RNA was then extracted and Viperin mRNA expression was analyzed by qRT-PCR. B Schematic representation of recombinant plasmids encoding different truncated ORF2 constructs. C LMH cells were transfected with plasmids expressing full-length ORF2 or the truncated domains S, P1, and P2 for 48 h, after which Viperin mRNA expression was quantified by qRT-PCR. D LMH cells were transfected with the indicated plasmids and then infected with GAstV-2 at MOI of 0.01. Viral copy numbers were measured by qRT-PCR at 48 hpi. In A, C, and D, data are presented as mean ± SD from three biological replicates. Student’s t-test was used to compare the empty plasmid group with the recombinant plasmid groups (*P < 0.05, **P < 0.01).

• 

  Figure 3. Viperin inhibits GAstV-2 infection in vitro. A, B Viral load in LMH cells transfected with increasing doses of pViperin followed by GAstV-2 infection (MOI = 0.01) were measured by qRT-PCR at 24 hpi (A) and 48 hpi (B). C, D Western blot analysis of GAstV-2 capsid protein expression in transfected and infected LMH cells at 24 hpi (C) and 48 hpi (D). E Efficiency of Viperin knockdown in LMH cells transfected with three distinct Viperin-specific siRNAs, measured by qRT-PCR at 48 hpi. F, G Viral loads in LMH cells transfected with siViperin-1 or siViperin-2 followed by GAstV-2 infection (MOI = 0.01), measured by qRT-PCR at 24 hpi (F) and 48 hpi (G). H Western blot analysis of GAstV-2 capsid protein levels in siRNA-transfected and virus-infected LMH cells at 24 and 48 hpi. In A–H, data are presented as mean ± SD from three biological replicates. Student’s t-test was used for comparison: in A–D, empty plasmid group versus increasing doses of the Viperin plasmid; in E–H, negative control siRNA group versus Viperin-specific siRNA groups (*P < 0.05, **P < 0.01).

• 

  Figure 4. Viperin restricts GAstV-2 replication at a post-entry stage. A Schematic diagram of the experimental design for assessing viral adsorption and internalization. B Viral adsorption analysis: LMH cells with or without Viperin overexpression were incubated with GAstV-2 (MOI = 1) at 4 °C for 1 h. Cell-associated viral RNA copies were quantified by qRT-PCR. C Viral internalization analysis: following adsorption at 4 °C, Viperin-overexpressing LMH cells were shifted to 37 °C for 1 h to allow viral entry, treated with 0.01% trypsin to remove noninternalized virus, and intracellular viral RNA copies were measured by qRT-PCR. (D–F) Protein-level analysis: GAstV-2 capsid protein levels were detected by Western blot under adsorption (D, F) and internalization (E, F) conditions, corresponding to the experimental schemes in B and C, respectively. In B–F, data are presented as mean ± SD from three biological replicates. Student’s t-test was used to compare the empty plasmid group with the Viperin plasmid group.

• 

  Figure 5. Viperin suppresses GAstV-2 RdRp activity. A Schematic representation of the cell-based reporter assay system for evaluating GAstV-2 RdRp activity. B RdRp activity in LMH cells transfected with pViperin or empty vector for 24 h, followed by GAstV-2 infection (MOI = 0.01) and treatment with varying NTP concentrations, as measured by the reporter assay system. C, D Viral load in transfected and infected LMH cells under different NTP conditions at 24 hpi (C) and 48 hpi (D), quantified by qRT-PCR. E, F GAstV-2 capsid protein expression in transfected and infected LMH cells at 24 hpi (E) and 48 hpi (F), detected by Western blot analysis. In B–F, data are presented as mean ± SD from three biological replicates. Student’s t-test was used to compare the empty plasmid group with the treated groups (*P < 0.05, **P < 0.01).

• 

  Figure 6. Viperin restricts GAstV-2 replication through ddhCTP production. A Quantification of intracellular ddhCTP levels in LMH cells following transfection with pViperin or empty vector control for 24 h, as determined by LC–MS analysis. B GAstV-2 RdRp activity in ddhCTP-treated and GAstV-2-infected LMH cells using a cell-based reporter assay system. C Viral load in ddhCTP-treated and GAstV-2-infected LMH cells at 36 hpi, measured by qRT-PCR at 36 hpi. D GAstV-2 capsid protein expression in ddhCTP-treated and GAstV-2-infected LMH cells at 36 hpi, detected by WB at 36 hpi. In A–D, data are presented as mean ± SD from three biological replicates. Student’s t-test was used for comparison: in A, empty plasmid group versus Viperin plasmid group; in B–D, GAstV-2-infected group versus ddhCTP-treated groups (*P < 0.05, **P < 0.01).

• 

  Figure 7. The radical SAM domain is essential for Viperin's antiviral function. A Schematic representation of Viperin domain architecture and mutant constructs. B GAstV-2 RdRp activity in LMH cells transfected with wild-type Viperin, Viperin mutants (ΔN, ΔC, DCA), or empty vector for 24 h, followed by infection with GAstV-2 (MOI = 0.01), assessed using a cell-based reporter assay. C, D Viral loads in transfected and infected LMH cells at 24 hpi (C) and 48 hpi (D), quantified by qRT-PCR. E–G GAstV-2 capsid protein expression in transfected and infected LMH cells at 24 hpi (E, G) and 48 hpi (F, G), measured by WB. H Intracellular ddhCTP concentrations in transfected and infected LMH cells, measured by LC–MS. In B–G, data are presented as mean ± SD from three biological replicates. Student’s t-test was used to compare the Viperin plasmid group with the empty plasmid group and with the Viperin mutant plasmid groups (*P < 0.05, **P < 0.01).

• 

  Figure 8. ddhCTP administration ameliorates GAstV-2 pathogenesis in goslings. A Survival rates of GAstV-2-infected goslings following intramuscular ddhCTP administration. B Body weight gain in infected goslings treated with ddhCTP or vehicle control at 7 dpi. C Relative kidney weight in different experimental groups at 7 dpi. D Representative gross morphological changes in different treatment groups. E Histopathological analysis of kidney, liver, and spleen sections from infected goslings with or without ddhCTP treatment. F Viral loads in kidney tissues quantified by qRT-PCR. G GAstV-2 capsid protein expression in kidney tissues detected by Western blot analysis. H The virus location was detected by immunohistochemistry and the mean optical density (MOD) of positive staining was calculated using Image-Pro Plus (I). In B, C, F, G, and I, data are presented as mean ± SD from ten gosling samples. Student’s t-test was used to compare the GAstV-2 group with the control group and with the ddhCTP group (*P < 0.05, **P < 0.01).

• 

  Figure 9. Proposed mechanism by which Viperin restricts goose astrovirus type 2 replication through inhibition of viral RdRP activity.