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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 5. RNA replication of CSFV occurs on the membranes of autophagosome-like vesicles. (A) PK-15 cells infected with CSFV (MOI = 1) for 48 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against the CD63 and CSFV proteins (E2 or NS5A), and the targeted protein staining was detected with secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, E2 and NS5A staining is shown in green (a and e), CD63 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 images are shown in (d and h) from the white-square frame-enclosed region in (c and g). Scale bar: 10 μm. One of 3 independent experiments is shown. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. One of 3 independent experiments is shown. (C) PK-15 (a and c) and 3D4/2 (b and d) cells were infected with CSFV at an MOI of 1 for 48 h. Subsequently, IEM analysis was performed using specific monoclonal antibodies against the CSFV NS5A and E2 proteins, and the targeted proteins were detected with a secondary antibody conjugated to 12-nm colloidal gold particles. The immunogold labeling showing the localization of CSFV proteins on the membranes of autophagosome-like vesicles is indicated by black arrows. Scale bar: 500 nm. One of 3 independent experiments is shown.
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Figure 5: Figure 5. RNA replication of CSFV occurs on the membranes of autophagosome-like vesicles. (A) PK-15 cells infected with CSFV (MOI = 1) for 48 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against the CD63 and CSFV proteins (E2 or NS5A), and the targeted protein staining was detected with secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, E2 and NS5A staining is shown in green (a and e), CD63 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 images are shown in (d and h) from the white-square frame-enclosed region in (c and g). Scale bar: 10 μm. One of 3 independent experiments is shown. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. One of 3 independent experiments is shown. (C) PK-15 (a and c) and 3D4/2 (b and d) cells were infected with CSFV at an MOI of 1 for 48 h. Subsequently, IEM analysis was performed using specific monoclonal antibodies against the CSFV NS5A and E2 proteins, and the targeted proteins were detected with a secondary antibody conjugated to 12-nm colloidal gold particles. The immunogold labeling showing the localization of CSFV proteins on the membranes of autophagosome-like vesicles is indicated by black arrows. Scale bar: 500 nm. One of 3 independent experiments is shown.

Mentions: CD63 is a widely used marker for lysosomes, and correlates well with the formation of autolysosomes.41 To confirm the relationship between autophagosome-like vesicles and viral replication, we examined the distribution of CD63 in virus-infected cells (Fig. 5A and B, b and f). We found that the CD63 protein colocalized with the E2 and NS5A proteins in CSFV-infected PK-15 and 3D4/2 cells (Fig. 5A and B, c and g), confirming that autophagosome-like vesicles are involved in the replication of CSFV and that the membranes of these structures could be required for CSFV 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 5. RNA replication of CSFV occurs on the membranes of autophagosome-like vesicles. (A) PK-15 cells infected with CSFV (MOI = 1) for 48 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against the CD63 and CSFV proteins (E2 or NS5A), and the targeted protein staining was detected with secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, E2 and NS5A staining is shown in green (a and e), CD63 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 images are shown in (d and h) from the white-square frame-enclosed region in (c and g). Scale bar: 10 μm. One of 3 independent experiments is shown. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. One of 3 independent experiments is shown. (C) PK-15 (a and c) and 3D4/2 (b and d) cells were infected with CSFV at an MOI of 1 for 48 h. Subsequently, IEM analysis was performed using specific monoclonal antibodies against the CSFV NS5A and E2 proteins, and the targeted proteins were detected with a secondary antibody conjugated to 12-nm colloidal gold particles. The immunogold labeling showing the localization of CSFV proteins on the membranes of autophagosome-like vesicles is indicated by black arrows. Scale bar: 500 nm. One of 3 independent experiments is shown.
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Figure 5: Figure 5. RNA replication of CSFV occurs on the membranes of autophagosome-like vesicles. (A) PK-15 cells infected with CSFV (MOI = 1) for 48 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against the CD63 and CSFV proteins (E2 or NS5A), and the targeted protein staining was detected with secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, E2 and NS5A staining is shown in green (a and e), CD63 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 images are shown in (d and h) from the white-square frame-enclosed region in (c and g). Scale bar: 10 μm. One of 3 independent experiments is shown. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. One of 3 independent experiments is shown. (C) PK-15 (a and c) and 3D4/2 (b and d) cells were infected with CSFV at an MOI of 1 for 48 h. Subsequently, IEM analysis was performed using specific monoclonal antibodies against the CSFV NS5A and E2 proteins, and the targeted proteins were detected with a secondary antibody conjugated to 12-nm colloidal gold particles. The immunogold labeling showing the localization of CSFV proteins on the membranes of autophagosome-like vesicles is indicated by black arrows. Scale bar: 500 nm. One of 3 independent experiments is shown.
Mentions: CD63 is a widely used marker for lysosomes, and correlates well with the formation of autolysosomes.41 To confirm the relationship between autophagosome-like vesicles and viral replication, we examined the distribution of CD63 in virus-infected cells (Fig. 5A and B, b and f). We found that the CD63 protein colocalized with the E2 and NS5A proteins in CSFV-infected PK-15 and 3D4/2 cells (Fig. 5A and B, c and g), confirming that autophagosome-like vesicles are involved in the replication of CSFV and that the membranes of these structures could be required for CSFV 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