Limits...
Autophagy enhances the replication of classical swine fever virus in vitro.

Pei J, Zhao M, Ye Z, Gou H, Wang J, Yi L, Dong X, Liu W, Luo Y, Liao M, Chen J - Autophagy (2013)

Bottom Line: However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed.We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy.Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine; South China Agricultural University; Guangzhou, China.

ABSTRACT
Autophagy plays an important role in cellular responses to pathogens. However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed. In this study, we showed that CSFV infection significantly increases the number of autophagy-like vesicles in the cytoplasm of host cells at the ultrastructural level. We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy. Meanwhile, high expression of ATG5 and BECN1 was detected in CSFV-infected cells; conversely, degradation of SQSTM1 was observed by immunoblotting, suggesting that CSFV infection triggered a complete autophagic response, most likely by the NS5A protein. Furthermore, by confocal immunofluorescence analysis, we discovered that both envelope protein E2 and nonstructural protein NS5A colocalized with LC3 and CD63 during CSFV infection. Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication. Finally, we demonstrated that alteration of cellular autophagy by autophagy regulators and shRNAs affects progeny virus production. Collectively, these findings provide strong evidence that CSFV infection needs an autophagy pathway to enhance viral replication and maturity in host cells.

Show MeSH

Related in: MedlinePlus

Figure 6. CSFV nonstructural protein NS5A induces autophagy in host cells. (A) PK-15 cells were transfected with vector pEGFP-N1 or plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein. At 48 hpi, cells were analyzed as described in the legend to Figure 4, but only with an antibody against LC3B and TRITC-conjugated goat anti-rabbit IgG. In the images, the expression of EGFP-NS5A is shown in green (a and e), LC3 staining is shown in red (b and f), and the signals of colocalization are shown in yellow in the merged images (c and g). The higher magnification image is shown in (d) from the white-square frame-enclosed region in (c). Scale bar: 10 μm. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (B) 3D4/2 cells were transfected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (C) PK-15 cells and 3D4/2 cells were transfected as described in (A), and mock-infected cells were used as the control. The cell samples were analyzed by immunoblotting with anti-LC3B and anti-ACTB (loading control) antibodies, and the efficiency of transfection was measured using an antibody against GFP. One of 3 independent experiments is shown. (D) PK-15 cells and 3D4/2 cells were transfected plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein for 24, 36, and 48 h. The cell samples were analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. One of 3 independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4389882&req=5

Figure 6: Figure 6. CSFV nonstructural protein NS5A induces autophagy in host cells. (A) PK-15 cells were transfected with vector pEGFP-N1 or plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein. At 48 hpi, cells were analyzed as described in the legend to Figure 4, but only with an antibody against LC3B and TRITC-conjugated goat anti-rabbit IgG. In the images, the expression of EGFP-NS5A is shown in green (a and e), LC3 staining is shown in red (b and f), and the signals of colocalization are shown in yellow in the merged images (c and g). The higher magnification image is shown in (d) from the white-square frame-enclosed region in (c). Scale bar: 10 μm. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (B) 3D4/2 cells were transfected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (C) PK-15 cells and 3D4/2 cells were transfected as described in (A), and mock-infected cells were used as the control. The cell samples were analyzed by immunoblotting with anti-LC3B and anti-ACTB (loading control) antibodies, and the efficiency of transfection was measured using an antibody against GFP. One of 3 independent experiments is shown. (D) PK-15 cells and 3D4/2 cells were transfected plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein for 24, 36, and 48 h. The cell samples were analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. One of 3 independent experiments is shown.

Mentions: NS5A is an important nonstructural protein of CSFV that is associated with virus replication. To further analyze whether CSFV replication is required for the induction of autophagy and determine the effect of NS5A on autophagy induction during CSFV infection, we expressed NS5A as a fusion protein with enhanced green fluorescent protein (EGFP) and transfected the cells with pEGFP-N1 as an empty vector control. We found that the fluorescence signals of LC3 and EGFP-NS5A were clearly enhanced in PK-15 and 3D4/2 cells after transfection with pEGFP-NS5A (Fig. 6A and B, a and b). Furthermore, the puncta accumulation and colocalization signals of the targeted proteins were detected by immunofluorescence analysis (Fig. 6A and B, c). In contrast, weak signals of LC3 and EGFP were observed in the control groups (Fig. 6A and B, e and f), and no colocalization signals were detected for the targeted proteins (Fig. 6A and B, g). Meanwhile, the average number of LC3 puncta in cells transfected with pEGFP-NS5A was significantly greater than in cells transfected with pEGFP-N1. Additionally, immunoblotting analysis was used to quantify the expression of LC3, SQSTM1, and NS5A. We found that pEGFP-NS5A significantly increased the level of LC3-II and promoted the degradation of SQSTM1 in host cells compared with pEGFP-N1-transfected and mock-infected cells (Fig. 6C and D). These data indicate that the replicase NS5A can induce autophagosome formation and activate a complete autophagic response.


Autophagy enhances the replication of classical swine fever virus in vitro.

Pei J, Zhao M, Ye Z, Gou H, Wang J, Yi L, Dong X, Liu W, Luo Y, Liao M, Chen J - Autophagy (2013)

Figure 6. CSFV nonstructural protein NS5A induces autophagy in host cells. (A) PK-15 cells were transfected with vector pEGFP-N1 or plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein. At 48 hpi, cells were analyzed as described in the legend to Figure 4, but only with an antibody against LC3B and TRITC-conjugated goat anti-rabbit IgG. In the images, the expression of EGFP-NS5A is shown in green (a and e), LC3 staining is shown in red (b and f), and the signals of colocalization are shown in yellow in the merged images (c and g). The higher magnification image is shown in (d) from the white-square frame-enclosed region in (c). Scale bar: 10 μm. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (B) 3D4/2 cells were transfected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (C) PK-15 cells and 3D4/2 cells were transfected as described in (A), and mock-infected cells were used as the control. The cell samples were analyzed by immunoblotting with anti-LC3B and anti-ACTB (loading control) antibodies, and the efficiency of transfection was measured using an antibody against GFP. One of 3 independent experiments is shown. (D) PK-15 cells and 3D4/2 cells were transfected plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein for 24, 36, and 48 h. The cell samples were analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. One of 3 independent experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389882&req=5

Figure 6: Figure 6. CSFV nonstructural protein NS5A induces autophagy in host cells. (A) PK-15 cells were transfected with vector pEGFP-N1 or plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein. At 48 hpi, cells were analyzed as described in the legend to Figure 4, but only with an antibody against LC3B and TRITC-conjugated goat anti-rabbit IgG. In the images, the expression of EGFP-NS5A is shown in green (a and e), LC3 staining is shown in red (b and f), and the signals of colocalization are shown in yellow in the merged images (c and g). The higher magnification image is shown in (d) from the white-square frame-enclosed region in (c). Scale bar: 10 μm. The average number of LC3 puncta in each cell was determined from at least 100 cells in each group. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (B) 3D4/2 cells were transfected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001. (C) PK-15 cells and 3D4/2 cells were transfected as described in (A), and mock-infected cells were used as the control. The cell samples were analyzed by immunoblotting with anti-LC3B and anti-ACTB (loading control) antibodies, and the efficiency of transfection was measured using an antibody against GFP. One of 3 independent experiments is shown. (D) PK-15 cells and 3D4/2 cells were transfected plasmid pEGFP-NS5A expressing the EGFP-NS5A fusion protein for 24, 36, and 48 h. The cell samples were analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. One of 3 independent experiments is shown.
Mentions: NS5A is an important nonstructural protein of CSFV that is associated with virus replication. To further analyze whether CSFV replication is required for the induction of autophagy and determine the effect of NS5A on autophagy induction during CSFV infection, we expressed NS5A as a fusion protein with enhanced green fluorescent protein (EGFP) and transfected the cells with pEGFP-N1 as an empty vector control. We found that the fluorescence signals of LC3 and EGFP-NS5A were clearly enhanced in PK-15 and 3D4/2 cells after transfection with pEGFP-NS5A (Fig. 6A and B, a and b). Furthermore, the puncta accumulation and colocalization signals of the targeted proteins were detected by immunofluorescence analysis (Fig. 6A and B, c). In contrast, weak signals of LC3 and EGFP were observed in the control groups (Fig. 6A and B, e and f), and no colocalization signals were detected for the targeted proteins (Fig. 6A and B, g). Meanwhile, the average number of LC3 puncta in cells transfected with pEGFP-NS5A was significantly greater than in cells transfected with pEGFP-N1. Additionally, immunoblotting analysis was used to quantify the expression of LC3, SQSTM1, and NS5A. We found that pEGFP-NS5A significantly increased the level of LC3-II and promoted the degradation of SQSTM1 in host cells compared with pEGFP-N1-transfected and mock-infected cells (Fig. 6C and D). These data indicate that the replicase NS5A can induce autophagosome formation and activate a complete autophagic response.

Bottom Line: However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed.We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy.Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine; South China Agricultural University; Guangzhou, China.

ABSTRACT
Autophagy plays an important role in cellular responses to pathogens. However, the impact of the autophagy machinery on classical swine fever virus (CSFV) infection is not yet confirmed. In this study, we showed that CSFV infection significantly increases the number of autophagy-like vesicles in the cytoplasm of host cells at the ultrastructural level. We also found the formation of 2 ubiquitin-like conjugation systems upon virus infection, including LC3-I/LC3-II conversion and ATG12-ATG5 conjugation, which are considered important indicators of autophagy. Meanwhile, high expression of ATG5 and BECN1 was detected in CSFV-infected cells; conversely, degradation of SQSTM1 was observed by immunoblotting, suggesting that CSFV infection triggered a complete autophagic response, most likely by the NS5A protein. Furthermore, by confocal immunofluorescence analysis, we discovered that both envelope protein E2 and nonstructural protein NS5A colocalized with LC3 and CD63 during CSFV infection. Examination by immunoelectron microscopy further confirmed the colocalization of both E2 and NS5A proteins with autophagosome-like vesicles, indicating that CSFV utilizes the membranes of these vesicles for replication. Finally, we demonstrated that alteration of cellular autophagy by autophagy regulators and shRNAs affects progeny virus production. Collectively, these findings provide strong evidence that CSFV infection needs an autophagy pathway to enhance viral replication and maturity in host cells.

Show MeSH
Related in: MedlinePlus