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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.

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Figure 8. Inhibition of autophagy with 3-MA reduces CSFV replication. (A and E) PK-15 (A) and 3D4/2 (E) cells were pretreated with 3-MA (5 mM) or DMSO (Control) for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the absence or presence of 3-MA (5 mM). At 24 and 48 hpi, cell samples were analyzed as described in the legend to Figure 7A and D. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were pretreated with 3-MA for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the presence of 3-MA (5 mM). At 48 hpi, the cells were analyzed with antibodies against LC3B and E2, as described in the legend to Figure 4. The fluorescence signals were visualized with confocal immunofluorescence microscopy. In the images, E2 staining is shown in green, LC3 staining is shown in red, and the signals of colocalization are shown in the merged images. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular copy numbers of CSFV were detected by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; **P < 0.01; ***P < 0.001. (D and H) PK-15 (D) and 3D4/2 (H) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using the immunofluorescence assay described in Materials and Methods. Results are expressed in units of TCID50/ml. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 8: Figure 8. Inhibition of autophagy with 3-MA reduces CSFV replication. (A and E) PK-15 (A) and 3D4/2 (E) cells were pretreated with 3-MA (5 mM) or DMSO (Control) for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the absence or presence of 3-MA (5 mM). At 24 and 48 hpi, cell samples were analyzed as described in the legend to Figure 7A and D. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were pretreated with 3-MA for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the presence of 3-MA (5 mM). At 48 hpi, the cells were analyzed with antibodies against LC3B and E2, as described in the legend to Figure 4. The fluorescence signals were visualized with confocal immunofluorescence microscopy. In the images, E2 staining is shown in green, LC3 staining is shown in red, and the signals of colocalization are shown in the merged images. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular copy numbers of CSFV were detected by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; **P < 0.01; ***P < 0.001. (D and H) PK-15 (D) and 3D4/2 (H) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using the immunofluorescence assay described in Materials and Methods. Results are expressed in units of TCID50/ml. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: To further determine the effect of autophagy on CSFV replication, we exposed the host cells to 3-MA, which inhibits autophagy by blocking the formation of autophagosomes,45,46 and analyzed the capability of CSFV replication by detecting viral envelope protein E2 expression and viral progeny yield by using relevant assays. As shown in Figure 8, 3-MA treatment reduced the expression of LC3-II and CSFV-E2 at 24 and 48 hpi in CSFV-infected PK-15 cells and decreased the virus copy number and titer (Fig. 8A, C, and D). Notably, the effect of 3-MA treatment on the extracellular viral load and titer was greater than the intracellular load and titer (Fig. 8C and D). Importantly, the LC3-positive puncta and the colocalization of LC3 and E2 decreased in the presence of 3-MA (Fig. 8B). Similar results for viral envelope protein E2 expression and viral progeny yield were obtained in treated 3D4/2 cells (Fig. 8E–H). Furthermore, because autophagosome formation can be inhibited by 3-MA, the decreased densitometry ratio of LC3-II (relative to ACTB) in 3-MA-treated cells further verified that the enhanced autophagy shown in Figures 1 and 2 was triggered by the CSFV infection. These findings suggest that the induction of autophagy during CSFV infection promotes virus replication.


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 8. Inhibition of autophagy with 3-MA reduces CSFV replication. (A and E) PK-15 (A) and 3D4/2 (E) cells were pretreated with 3-MA (5 mM) or DMSO (Control) for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the absence or presence of 3-MA (5 mM). At 24 and 48 hpi, cell samples were analyzed as described in the legend to Figure 7A and D. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were pretreated with 3-MA for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the presence of 3-MA (5 mM). At 48 hpi, the cells were analyzed with antibodies against LC3B and E2, as described in the legend to Figure 4. The fluorescence signals were visualized with confocal immunofluorescence microscopy. In the images, E2 staining is shown in green, LC3 staining is shown in red, and the signals of colocalization are shown in the merged images. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular copy numbers of CSFV were detected by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; **P < 0.01; ***P < 0.001. (D and H) PK-15 (D) and 3D4/2 (H) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using the immunofluorescence assay described in Materials and Methods. Results are expressed in units of TCID50/ml. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 8: Figure 8. Inhibition of autophagy with 3-MA reduces CSFV replication. (A and E) PK-15 (A) and 3D4/2 (E) cells were pretreated with 3-MA (5 mM) or DMSO (Control) for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the absence or presence of 3-MA (5 mM). At 24 and 48 hpi, cell samples were analyzed as described in the legend to Figure 7A and D. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were pretreated with 3-MA for 4 h. After 1 h of virus absorption at an MOI of 0.5, the cells were further cultured in fresh medium in the presence of 3-MA (5 mM). At 48 hpi, the cells were analyzed with antibodies against LC3B and E2, as described in the legend to Figure 4. The fluorescence signals were visualized with confocal immunofluorescence microscopy. In the images, E2 staining is shown in green, LC3 staining is shown in red, and the signals of colocalization are shown in the merged images. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular copy numbers of CSFV were detected by qRT-PCR. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; **P < 0.01; ***P < 0.001. (D and H) PK-15 (D) and 3D4/2 (H) cells were pretreated and infected as described in (A and E). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using the immunofluorescence assay described in Materials and Methods. Results are expressed in units of TCID50/ml. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: To further determine the effect of autophagy on CSFV replication, we exposed the host cells to 3-MA, which inhibits autophagy by blocking the formation of autophagosomes,45,46 and analyzed the capability of CSFV replication by detecting viral envelope protein E2 expression and viral progeny yield by using relevant assays. As shown in Figure 8, 3-MA treatment reduced the expression of LC3-II and CSFV-E2 at 24 and 48 hpi in CSFV-infected PK-15 cells and decreased the virus copy number and titer (Fig. 8A, C, and D). Notably, the effect of 3-MA treatment on the extracellular viral load and titer was greater than the intracellular load and titer (Fig. 8C and D). Importantly, the LC3-positive puncta and the colocalization of LC3 and E2 decreased in the presence of 3-MA (Fig. 8B). Similar results for viral envelope protein E2 expression and viral progeny yield were obtained in treated 3D4/2 cells (Fig. 8E–H). Furthermore, because autophagosome formation can be inhibited by 3-MA, the decreased densitometry ratio of LC3-II (relative to ACTB) in 3-MA-treated cells further verified that the enhanced autophagy shown in Figures 1 and 2 was triggered by the CSFV infection. These findings suggest that the induction of autophagy during CSFV infection promotes virus replication.

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