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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 4. CSFV infection induces the redistribution of the autophagy marker LC3 in host cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) or treated with rapamycin (Rapa, 100 nM) for 48 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the CSFV proteins (E2 or NS5A), followed by the corresponding secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The cell nucleus were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, the nucleus staining is shown in blue (a and f), E2 and NS5A staining is shown in green (b and g), LC3 staining is shown in red (c and h), and the signals of colocalization are shown in yellow in merged images (d and i). The higher magnification images are shown in (e and j) from the white-square frame-enclosed region in (d and i). The mock- and UV-CSFV-infected cells as well as the rapamycin treatment group were analyzed with anti-LC3B antibodies and used as the control groups (k–m). 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 < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.
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Figure 4: Figure 4. CSFV infection induces the redistribution of the autophagy marker LC3 in host cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) or treated with rapamycin (Rapa, 100 nM) for 48 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the CSFV proteins (E2 or NS5A), followed by the corresponding secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The cell nucleus were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, the nucleus staining is shown in blue (a and f), E2 and NS5A staining is shown in green (b and g), LC3 staining is shown in red (c and h), and the signals of colocalization are shown in yellow in merged images (d and i). The higher magnification images are shown in (e and j) from the white-square frame-enclosed region in (d and i). The mock- and UV-CSFV-infected cells as well as the rapamycin treatment group were analyzed with anti-LC3B antibodies and used as the control groups (k–m). 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 < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.

Mentions: To verify the correlation between the autophagy and viral replication during the progression of CSFV infection, we first detected the subcellular localizations of viral proteins and LC3 in PK-15 and 3D4/2 cells that had been infected with CSFV, by using immunofluorescence analysis with antibodies specific to E2, NS5A, and LC3B. As illustrated in Figure 4A and B, significant fluorescence signals corresponding to the LC3 and CSFV proteins were detected in CSFV-infected PK-15 and 3D4/2 cells, with puncta accumulation (Fig. 4A and B, b and c, g and h). The fluorescence puncta of both E2 and NS5A were highly colocalized with the fluorescence puncta of LC3 (Fig. 4A and B, d and i). In contrast, the LC3 puncta accumulation was not observed in mock- and UV-CSFV-infected cells (Fig. 4A and B, k and m), indicating that UV-inactivated CSFV had lost its capability of inducing the formation of the autophagosome, consistent with the data shown in Figure 2. Additionally, the average number of LC3 puncta in the infected cells was significantly greater than that in rapamycin-treated cells (Fig. 4A and B, lower right part). These results indicate that CSFV infection induces the redistribution of autophagy marker LC3 in host cells, and the autophagosome could be involved in viral 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 4. CSFV infection induces the redistribution of the autophagy marker LC3 in host cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) or treated with rapamycin (Rapa, 100 nM) for 48 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the CSFV proteins (E2 or NS5A), followed by the corresponding secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The cell nucleus were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, the nucleus staining is shown in blue (a and f), E2 and NS5A staining is shown in green (b and g), LC3 staining is shown in red (c and h), and the signals of colocalization are shown in yellow in merged images (d and i). The higher magnification images are shown in (e and j) from the white-square frame-enclosed region in (d and i). The mock- and UV-CSFV-infected cells as well as the rapamycin treatment group were analyzed with anti-LC3B antibodies and used as the control groups (k–m). 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 < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.
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Figure 4: Figure 4. CSFV infection induces the redistribution of the autophagy marker LC3 in host cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) or treated with rapamycin (Rapa, 100 nM) for 48 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against LC3B and the CSFV proteins (E2 or NS5A), followed by the corresponding secondary antibodies conjugated to FITC and TRITC as described in Materials and Methods. The cell nucleus were counterstained with DAPI. The fluorescence signals were visualized by confocal immunofluorescence microscopy. In the images, the nucleus staining is shown in blue (a and f), E2 and NS5A staining is shown in green (b and g), LC3 staining is shown in red (c and h), and the signals of colocalization are shown in yellow in merged images (d and i). The higher magnification images are shown in (e and j) from the white-square frame-enclosed region in (d and i). The mock- and UV-CSFV-infected cells as well as the rapamycin treatment group were analyzed with anti-LC3B antibodies and used as the control groups (k–m). 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 < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05. (B) 3D4/2 cells were infected and analyzed as in (A). Scale bar: 10 μm. The data represent the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; #P > 0.05.
Mentions: To verify the correlation between the autophagy and viral replication during the progression of CSFV infection, we first detected the subcellular localizations of viral proteins and LC3 in PK-15 and 3D4/2 cells that had been infected with CSFV, by using immunofluorescence analysis with antibodies specific to E2, NS5A, and LC3B. As illustrated in Figure 4A and B, significant fluorescence signals corresponding to the LC3 and CSFV proteins were detected in CSFV-infected PK-15 and 3D4/2 cells, with puncta accumulation (Fig. 4A and B, b and c, g and h). The fluorescence puncta of both E2 and NS5A were highly colocalized with the fluorescence puncta of LC3 (Fig. 4A and B, d and i). In contrast, the LC3 puncta accumulation was not observed in mock- and UV-CSFV-infected cells (Fig. 4A and B, k and m), indicating that UV-inactivated CSFV had lost its capability of inducing the formation of the autophagosome, consistent with the data shown in Figure 2. Additionally, the average number of LC3 puncta in the infected cells was significantly greater than that in rapamycin-treated cells (Fig. 4A and B, lower right part). These results indicate that CSFV infection induces the redistribution of autophagy marker LC3 in host cells, and the autophagosome could be involved in viral 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