The inhibitory effect of phosphorylated Codonopsis pilosula polysaccharide on autophagosomes formation contributes to the inhibition of duck hepatitis A virus replication

Abstract

Duck hepatitis A virus type 1 (DHAV) infection causes duck viral hepatitis and results in enormous loss to poultry farming industry. We reported that phosphorylated Codonopsis pilosula polysaccharide (pCPPS) inhibited DHAV genome replication. Here we further explored its underlying antiviral mechanisms. Autophagosomes formation is essential for the genome replication of picornaviruses. In this study, Western blot, confocal microscopy observation, and ELISA methods were performed to analyze polysaccharides' effects on autophagy by the in vitro and in vivo experiments. Results obtained from in vitro and in vivo experiments showed that Codonopsis pilosula polysaccharide did not play a role in regulating autophagy and had no therapeutic effects on infected ducklings. However, pCPPS treatment downregulated LC3-II expression level activated by DHAV and rapamycin, indicating the inhibition of autophagosomes formation. The interdiction of autophagosomes formation resulted in the inhibition of DHAV genome replication. Further study showed that pCPPS treatment reduced the concentration of phosphatidylinositol-3-phosphate (PI3P), an important component of membrane, in cells and serum, and consequently, autophagosomes formation was downregulated. In vivo experiments also verified the therapeutic effect of pCPPS. Phosphorylated Codonopsis pilosula polysaccharide treatment increased the infected ducklings' survival rate and alleviated hepatic injury. Our studies verified the effects of pCPPS against DHAV infection in duck embryo hepatocytes and ducklings and confirmed that phosphorylated modification enhanced the bioactivities of polysaccharides. The results also stated pCPPS's antiviral mechanisms, provided fundamental basis for the development of new anti-DHAV agents.

Keywords: antiviral; autophagy; duck hepatitis A virus; phosphorylated polysaccharide.

Figure 1

The polysaccharides' effects on duck hepatitis A virus (DHAV)-induced autophagy. Cells were infected with or without DHAV and then treated with Codonopsis pilosula polysaccharide (CPPS) or phosphorylated C. pilosula polysaccharide (pCPPS). (A) Western blot analysis of LC3-II protein. β-actin was selected as a protein loading control. (B) The ratio of LC3-II/β-actin. The decrease of the ratio represents the inhibition of autophagy. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (***P < 0.001; ns, no significance).

Figure 2

The polysaccharides' effects on rapamycin-induced autophagy. Cells were treated with rapamycin (Rapa) or polysaccharides (Codonopsis pilosula polysaccharide [CPPS] and phosphorylated Codonopsis pilosula polysaccharide [pCPPS]). (A) The localization of LC3 protein following diverse treatment. (B) Western blot analysis of LC3-II expression. β-actin was selected as a protein loading control. (C) The ratio of LC3-II/β-actin. The increase of the ratio represents autophagy. Results were shown as means ± SD (n = 3). Significance was analyzed with ANOVA (****P < 0.0001; ns, no significance). (D) Western blot analysis of p62 protein. Its decrease represents the fusion of autophagosome with lysosome. β-actin was selected as a protein loading control. (E) The ratio of p62/β-actin. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (***P < 0.001; ns, no significance).

Figure 3

Polysaccharides' effects on the concentration of PI3P in duck embryo hepatocyte (DEHs). Infected or non-infected cells were treated with polysaccharides (Codonopsis pilosula polysaccharide [CPPS] and phosphorylated Codonopsis pilosula polysaccharide [pCPPS]) or wortmannin (Wort). (A) The concentration of PI3P in infected DEHs. Results were shown as means ± SD (n = 3). Significance was analyzed with ANOVA (*P < 0.05; ****P < 0.0001). (B) The concentration of PI3P in non-infected DEHs. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (****P < 0.0001).

Figure 4

The LC3-II expression levels in tissues of ducklings infected with duck hepatitis A virus type 1 (DHAV). (A) Western blot analysis of LC3-II expression. β-actin was selected as a protein loading control. (B) The ratio of LC3-II/β-actin. The increase of the ratio represents autophagy. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (*P < 0.05; **P < 0.01; ***P < 0.0001; ns, no significance).

Figure 5

The LC3-II expression levels in tissues of ducklings treated with rapamycin. (A) Western blot analysis of LC3-II expression. β-actin was selected as a protein loading control. (B) The ratio of LC3-II/β-actin. The increase of the ratio represents autophagy. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (*P < 0.05; **P < 0.01; ns, no significance).

Figure 6

Concentration of PI3P in serum. Results are shown as means ± SD (n = 3). Significance was analyzed with ANOVA (*P < 0.05; ns, no significance).

Figure 7

The evaluation of polysaccharides' therapeutic effects. (A) Survival rates of ducklings that were treated with Codonopsis pilosula polysaccharide (CPPS), phosphorylated Codonopsis pilosula polysaccharide (pCPPS), or normal saline after being infected with duck hepatitis A virus type 1 (DHAV) (n = 10). (B) Visual pathological changes of livers in different groups.