A viroporin-like 2B protein of duck hepatitis A virus 1 that induces incomplete autophagy in DEF cells

Figures

• 

  Figure 1. Prediction of viroporin motifs in 2B Protein. (A) Transmembrane prediction. Blue baseline represents the fragment toward the intramembrane, pink baseline represents the fragment toward the outside of the membrane, and red baseline represents the transmembrane region. (B) Kyte-Doolittle hydropathy plots. Blue lines denote hydropathy and red lines denote amphipathicity. (C) PSIPred secondary structure algorithms prediction.

• 

  Figure 2. Expression analysis of eukaryotic recombinant plasmid. (A) The pCAGGS-2B-HA plasmid (4.0 μg) were transfected into duck embryo fibroblasts (DEF) cells. Cell lysates were collected at 48-h post-transfection and analyzed by immunoblot using indicated antibodies. (B) The pCAGGS-Flag-2B plasmid (4.0 μg) was transfected into DEF cells. Cell lysates were collected at 48-h post-transfection and analyzed by immunoblot using indicated antibodies.

• 

  Figure 3. Membrane topology of the 2B protein in Duck embryo fibroblasts (DEF) cells. The pCAGGS-2B-HA (4.0 μg) or pCAGGS-Flag-2B plasmid (4.0 μg) were transfected into DEF cells. The Digitonin group was permeabilized with digitalis saponin (0.01 mg / mL); the Triton group was permeabilized with 0.25% Triton-100. The blue fluorescence represents the nucleus and the red fluorescence represents the target protein. Images were captured by fluorescence microscopy using a 40× objective.

• 

  Figure 4. Effect of 2B protein on cytosolic calcium concentration in host cells. The pCAGGS-2B-HA (4.0 μg) or pCAGGS plasmid (4.0 μg) were transfected into duck embryo fibroblasts (DEF) cells. Then the cells were stained with Fluo-3 AM at 48-h post-transfection. A histogram was constructed to reveal the changes in the intracellular Ca2+concentration. Differences between two groups were analyzed using Student's t test and were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001.

• 

  Figure 5. Autophagy induced by the 2B protein in Duck embryo fibroblasts (DEF) cells. (A, C) The GFP-LC3 plasmid (2.0 μg) was transfected into DEF cells. One group was cotransfected with 2.0 μg of pCAGGS-2B-HA, another group was cotransfected with 2.0 μg of the pCAGGS empty vector as a negative control, and the last group was added with rapamycin (100 nM) 4 h before harvest as a positive control. (A) The microtubule-associated protein 1 light chain 3 II (LC3-II) levels were determined by Western blot analysis with an anti-LC3 antibody. (B) Band intensity was quantified using ImageJ, and β-actin was used as a loading control. The ratio of LC3-II to β-actin was normalized to the pCAGGS empty vector group. Differences between two groups were analyzed using Student's t test and were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001. (C) Cell slides were collected at 48-h post-transfection and stained with 4′,6-diamidino-2-phenylindole (DAPI) after fixation and permeation. The results were observed with a fluorescence microscope after glycerol fixation. Blue fluorescence represents the nucleus, green fluorescence represents autophagosomes, and red fluorescence represents the 2B…

• 

  Figure 6. Effect of 2B protein on autophagic flow. (A, D) The GFP-LC3 plasmid (2.0 μg) was transfected into duck embryo fibroblasts (DEF) cells. The rapamycin and starve groups were used as positive controls, and the pCAGGS empty group was used as negative controls. (A) One group was cotransfected with 2.0 μg of pCAGGS-2B-HA, another group was cotransfected with 2.0 μg of the pCAGGS empty vector and the p62 protein levels were determined by Western blot analysis with an anti-SQSTM1/p62 antibody. (B) Band intensity was quantified using ImageJ, and β-actin was used as a loading control. The ratio of SQSTM1/p62 to β-actin was normalized to the pCAGGS-2B-HA group. Differences between two groups were analyzed using Student's t-test and were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001. (C) Double fluorescence mRFP-GFP-LC3 indicator system to detect whether autophagosomes are fused to lysosomes. Images were captured by fluorescence microscopy using a 40× objective. (D) Lysosomal staining to detect colocalization of autophagosomes and lysosomes. Blue fluorescence represents the nucleus, green fluorescence represents the LC3 protein, and red fluorescence represents lysosomes.…