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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 10. Inhibition of autophagy with LC3B-targeting shRNA reduces the replication of CSFV. (A and E) PK-15 (A) and 3D4/2 (E) cells were transfected with shRNAs targeting LC3B or scrambled shRNAs for 48 h, followed by mock infection and CSFV infection at an MOI of 0.5. At 24 hpi, the silencing efficiency of LC3B shRNA, as well as the expression of autophagy marker proteins and CSFV-E2 were analyzed as described in the legend to Figure 2A. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were transfected with LC3B shRNA for 48 h, followed by CSFV infection at an MOI of 0.5. At 24 hpi, the cells were analyzed as described in the legend to Figure 8B and F. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were transfected as described in (A and E), followed by CSFV infection at an MOI of 0.5 for 24 h. 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 transfected and infected as described in (C and G). At 24 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 10: Figure 10. Inhibition of autophagy with LC3B-targeting shRNA reduces the replication of CSFV. (A and E) PK-15 (A) and 3D4/2 (E) cells were transfected with shRNAs targeting LC3B or scrambled shRNAs for 48 h, followed by mock infection and CSFV infection at an MOI of 0.5. At 24 hpi, the silencing efficiency of LC3B shRNA, as well as the expression of autophagy marker proteins and CSFV-E2 were analyzed as described in the legend to Figure 2A. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were transfected with LC3B shRNA for 48 h, followed by CSFV infection at an MOI of 0.5. At 24 hpi, the cells were analyzed as described in the legend to Figure 8B and F. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were transfected as described in (A and E), followed by CSFV infection at an MOI of 0.5 for 24 h. 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 transfected and infected as described in (C and G). At 24 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: Our results using pharmacological regulators highlighted the crucial role of the autophagy machinery in CSFV replication. To further explore the relationship between the autophagy machinery and virus replication, shRNA knockdown experiments were performed to specifically deplete endogenous BECN1 and MAP1LC3B (LC3B) proteins. As shown in Figures 9 and 10, PK-15 and 3D4/2 cells transfected with shBECN1 and shLC3B presented significantly decreased levels of endogenous BECN1 and LC3 proteins compared with cells transfected with nontargeting (scrambled) shRNAs comprising the control group (Figs. 9 and 10, A and E). Importantly, suppression of BECN1 and LC3 expression strongly reduced the expression of viral envelope protein E2 and the viral progeny yield in CSFV-infected PK-15 cells compared with the control group (Figs. 9 and 10, A, C, and D). Similar results were also obtained in infected 3D4/2 cells (Figs. 9 and 10, E, G, and H). Notably, the LC3-positive puncta and the colocalization of LC3 and E2 disappeared when depleting endogenous BECN1 and LC3 in both PK-15 and 3D4/2 cells (Figs. 9 and 10, B and F). These data further reveal that autophagy plays an important role in the replication of CSFV.


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 10. Inhibition of autophagy with LC3B-targeting shRNA reduces the replication of CSFV. (A and E) PK-15 (A) and 3D4/2 (E) cells were transfected with shRNAs targeting LC3B or scrambled shRNAs for 48 h, followed by mock infection and CSFV infection at an MOI of 0.5. At 24 hpi, the silencing efficiency of LC3B shRNA, as well as the expression of autophagy marker proteins and CSFV-E2 were analyzed as described in the legend to Figure 2A. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were transfected with LC3B shRNA for 48 h, followed by CSFV infection at an MOI of 0.5. At 24 hpi, the cells were analyzed as described in the legend to Figure 8B and F. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were transfected as described in (A and E), followed by CSFV infection at an MOI of 0.5 for 24 h. 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 transfected and infected as described in (C and G). At 24 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 10: Figure 10. Inhibition of autophagy with LC3B-targeting shRNA reduces the replication of CSFV. (A and E) PK-15 (A) and 3D4/2 (E) cells were transfected with shRNAs targeting LC3B or scrambled shRNAs for 48 h, followed by mock infection and CSFV infection at an MOI of 0.5. At 24 hpi, the silencing efficiency of LC3B shRNA, as well as the expression of autophagy marker proteins and CSFV-E2 were analyzed as described in the legend to Figure 2A. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; ***P < 0.001. (B and F) PK-15 (B) and 3D4/2 (F) cells were transfected with LC3B shRNA for 48 h, followed by CSFV infection at an MOI of 0.5. At 24 hpi, the cells were analyzed as described in the legend to Figure 8B and F. Scale bar: 10 μm. One of 3 independent experiments is shown. (C and G) PK-15 (C) and 3D4/2 (G) cells were transfected as described in (A and E), followed by CSFV infection at an MOI of 0.5 for 24 h. 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 transfected and infected as described in (C and G). At 24 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: Our results using pharmacological regulators highlighted the crucial role of the autophagy machinery in CSFV replication. To further explore the relationship between the autophagy machinery and virus replication, shRNA knockdown experiments were performed to specifically deplete endogenous BECN1 and MAP1LC3B (LC3B) proteins. As shown in Figures 9 and 10, PK-15 and 3D4/2 cells transfected with shBECN1 and shLC3B presented significantly decreased levels of endogenous BECN1 and LC3 proteins compared with cells transfected with nontargeting (scrambled) shRNAs comprising the control group (Figs. 9 and 10, A and E). Importantly, suppression of BECN1 and LC3 expression strongly reduced the expression of viral envelope protein E2 and the viral progeny yield in CSFV-infected PK-15 cells compared with the control group (Figs. 9 and 10, A, C, and D). Similar results were also obtained in infected 3D4/2 cells (Figs. 9 and 10, E, G, and H). Notably, the LC3-positive puncta and the colocalization of LC3 and E2 disappeared when depleting endogenous BECN1 and LC3 in both PK-15 and 3D4/2 cells (Figs. 9 and 10, B and F). These data further reveal that autophagy plays an important role in the replication of CSFV.

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