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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 1. CSFV infection increases the formation of autophagosome-like vesicles. (A) PK-15 (a–c) and 3D4/2 (d–f) cells were mock-infected (a and d) or infected with CSFV (b and e) at an MOI of 1 for 48 h and studied by electron microscopy. White arrows indicate the structures with the characteristics of autophagosomes (b and e). The cells were also processed for IEM analysis. LC3 was visualized with specific antibodies and detected with a secondary antibody conjugated to 18-nm colloidal gold particles. LC3 protein immunogold labeling of CSFV-infected cells is shown in (c and f). The immunogold labeling localized to the infection-associated membranes is indicated by black arrows in the infected cells. Scale bar: 500 nm. (B) Quantification of the autophagosome-like vesicles per cell image. Average number of the vesicles in each cell was obtained from at least 10 cells undergoing each treatment. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001.
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Figure 1: Figure 1. CSFV infection increases the formation of autophagosome-like vesicles. (A) PK-15 (a–c) and 3D4/2 (d–f) cells were mock-infected (a and d) or infected with CSFV (b and e) at an MOI of 1 for 48 h and studied by electron microscopy. White arrows indicate the structures with the characteristics of autophagosomes (b and e). The cells were also processed for IEM analysis. LC3 was visualized with specific antibodies and detected with a secondary antibody conjugated to 18-nm colloidal gold particles. LC3 protein immunogold labeling of CSFV-infected cells is shown in (c and f). The immunogold labeling localized to the infection-associated membranes is indicated by black arrows in the infected cells. Scale bar: 500 nm. (B) Quantification of the autophagosome-like vesicles per cell image. Average number of the vesicles in each cell was obtained from at least 10 cells undergoing each treatment. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001.

Mentions: To determine whether CSFV infection regulates cellular autophagy, we examined the formation of autophagosome-like vesicles in CSFV-infected PK-15 and 3D4/2 cells by using transmission electron microscopy (TEM) and quantitative analysis. We found that the number of double- or single-membrane vesicles increased in the cytoplasm of CSFV-infected PK-15 cells (Fig. 1A, b). Similar results were obtained in CSFV-infected 3D4/2 cells (Fig. 1A, e). Compared with CSFV-infected cells, autophagosome-like vesicles were rarely seen in the mock-infected cells (Fig. 1A, a and d). Quantitative analysis also showed a significant increase in the quantity of single- and double-membrane vesicles in the CSFV-infected cells compared with the uninfected cells (Fig. 1B). To determine whether the formation of double-membrane vesicles is linked to autophagy during CSFV infection, immunoelectron microscopy (IEM) was performed on CSFV-infected cells. The results showed the expression of autophagy protein microtubule-associated protein 1 light chain 3 (LC3) during virus infection, and the immunogold labeling localized the infection-associated membranes (Fig. 1A, c and f). The negative controls, which were incubated with normal rabbit immunoglobulin G and without the primary antibody, showed no positive signals (data not shown). These results show that CSFV infection can induce autophagosome formation in vitro.


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 1. CSFV infection increases the formation of autophagosome-like vesicles. (A) PK-15 (a–c) and 3D4/2 (d–f) cells were mock-infected (a and d) or infected with CSFV (b and e) at an MOI of 1 for 48 h and studied by electron microscopy. White arrows indicate the structures with the characteristics of autophagosomes (b and e). The cells were also processed for IEM analysis. LC3 was visualized with specific antibodies and detected with a secondary antibody conjugated to 18-nm colloidal gold particles. LC3 protein immunogold labeling of CSFV-infected cells is shown in (c and f). The immunogold labeling localized to the infection-associated membranes is indicated by black arrows in the infected cells. Scale bar: 500 nm. (B) Quantification of the autophagosome-like vesicles per cell image. Average number of the vesicles in each cell was obtained from at least 10 cells undergoing each treatment. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001.
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Figure 1: Figure 1. CSFV infection increases the formation of autophagosome-like vesicles. (A) PK-15 (a–c) and 3D4/2 (d–f) cells were mock-infected (a and d) or infected with CSFV (b and e) at an MOI of 1 for 48 h and studied by electron microscopy. White arrows indicate the structures with the characteristics of autophagosomes (b and e). The cells were also processed for IEM analysis. LC3 was visualized with specific antibodies and detected with a secondary antibody conjugated to 18-nm colloidal gold particles. LC3 protein immunogold labeling of CSFV-infected cells is shown in (c and f). The immunogold labeling localized to the infection-associated membranes is indicated by black arrows in the infected cells. Scale bar: 500 nm. (B) Quantification of the autophagosome-like vesicles per cell image. Average number of the vesicles in each cell was obtained from at least 10 cells undergoing each treatment. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; ***P < 0.001.
Mentions: To determine whether CSFV infection regulates cellular autophagy, we examined the formation of autophagosome-like vesicles in CSFV-infected PK-15 and 3D4/2 cells by using transmission electron microscopy (TEM) and quantitative analysis. We found that the number of double- or single-membrane vesicles increased in the cytoplasm of CSFV-infected PK-15 cells (Fig. 1A, b). Similar results were obtained in CSFV-infected 3D4/2 cells (Fig. 1A, e). Compared with CSFV-infected cells, autophagosome-like vesicles were rarely seen in the mock-infected cells (Fig. 1A, a and d). Quantitative analysis also showed a significant increase in the quantity of single- and double-membrane vesicles in the CSFV-infected cells compared with the uninfected cells (Fig. 1B). To determine whether the formation of double-membrane vesicles is linked to autophagy during CSFV infection, immunoelectron microscopy (IEM) was performed on CSFV-infected cells. The results showed the expression of autophagy protein microtubule-associated protein 1 light chain 3 (LC3) during virus infection, and the immunogold labeling localized the infection-associated membranes (Fig. 1A, c and f). The negative controls, which were incubated with normal rabbit immunoglobulin G and without the primary antibody, showed no positive signals (data not shown). These results show that CSFV infection can induce autophagosome formation in vitro.

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