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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 3. CSFV infection enhances autophagic flux. (A and B) PK-15 (A) and 3D4/2 (B) cells were mock-infected or infected with CSFV (MOI = 1) for 12, 24, and 48 h. The cell samples were then analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. (C and D) PK-15 (C) and 3D4/2 (D) cells were infected with CSFV (MOI = 1) in the presence or absence of E64d (10 μg/ml). At 24 and 48 hpi, cell lysates were prepared and analyzed by immunoblotting using anti-LC3B, anti-SQSTM1, anti-E2, and anti-ACTB (for detection of ACTB as loading control) antibodies. The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to the ACTB control. 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.
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Figure 3: Figure 3. CSFV infection enhances autophagic flux. (A and B) PK-15 (A) and 3D4/2 (B) cells were mock-infected or infected with CSFV (MOI = 1) for 12, 24, and 48 h. The cell samples were then analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. (C and D) PK-15 (C) and 3D4/2 (D) cells were infected with CSFV (MOI = 1) in the presence or absence of E64d (10 μg/ml). At 24 and 48 hpi, cell lysates were prepared and analyzed by immunoblotting using anti-LC3B, anti-SQSTM1, anti-E2, and anti-ACTB (for detection of ACTB as loading control) antibodies. The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to the ACTB control. 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.

Mentions: To determine whether a complete autophagic response is triggered by CSFV infection, we first measured the degradation of SQSTM1, a marker for autophagy-mediated protein degradation pathway, by using immunoblotting analysis.33,37-39 As shown in Figure 3A and B, CSFV-infected cells had decreased levels of the SQSTM1 protein, from 12 to 48 h post-infection (hpi) (Fig. 3A and B, lanes 2, 4, and 6). In contrast, the level of SQSTM1 protein in mock-infected cells was higher than that in infected cells (Fig. 3A and B, lanes 1, 3, and 5). To confirm autophagic flux with viral infection, the levels of LC3-II and SQSTM1 were determined following treatment with or without the protease inhibitor E64d in infected cells, a widely used method for assessing autophagic flux.33,39,40 As shown in Figure 3C and D, E64d increased the levels of LC3-II and SQSTM1 in both PK-15 and 3D4/2 cells, at 24 and 48 hpi, compared with control groups. Interestingly, E64d treatment also increased the level of the E2 protein during CSFV infection (Fig. 3C and D). These data indicate that a complete autophagic response can be induced in host cells following CSFV infection.


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 3. CSFV infection enhances autophagic flux. (A and B) PK-15 (A) and 3D4/2 (B) cells were mock-infected or infected with CSFV (MOI = 1) for 12, 24, and 48 h. The cell samples were then analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. (C and D) PK-15 (C) and 3D4/2 (D) cells were infected with CSFV (MOI = 1) in the presence or absence of E64d (10 μg/ml). At 24 and 48 hpi, cell lysates were prepared and analyzed by immunoblotting using anti-LC3B, anti-SQSTM1, anti-E2, and anti-ACTB (for detection of ACTB as loading control) antibodies. The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to the ACTB control. 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.
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Figure 3: Figure 3. CSFV infection enhances autophagic flux. (A and B) PK-15 (A) and 3D4/2 (B) cells were mock-infected or infected with CSFV (MOI = 1) for 12, 24, and 48 h. The cell samples were then analyzed by immunoblotting with anti-SQSTM1 and anti-ACTB (loading control) antibodies. (C and D) PK-15 (C) and 3D4/2 (D) cells were infected with CSFV (MOI = 1) in the presence or absence of E64d (10 μg/ml). At 24 and 48 hpi, cell lysates were prepared and analyzed by immunoblotting using anti-LC3B, anti-SQSTM1, anti-E2, and anti-ACTB (for detection of ACTB as loading control) antibodies. The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to the ACTB control. 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.
Mentions: To determine whether a complete autophagic response is triggered by CSFV infection, we first measured the degradation of SQSTM1, a marker for autophagy-mediated protein degradation pathway, by using immunoblotting analysis.33,37-39 As shown in Figure 3A and B, CSFV-infected cells had decreased levels of the SQSTM1 protein, from 12 to 48 h post-infection (hpi) (Fig. 3A and B, lanes 2, 4, and 6). In contrast, the level of SQSTM1 protein in mock-infected cells was higher than that in infected cells (Fig. 3A and B, lanes 1, 3, and 5). To confirm autophagic flux with viral infection, the levels of LC3-II and SQSTM1 were determined following treatment with or without the protease inhibitor E64d in infected cells, a widely used method for assessing autophagic flux.33,39,40 As shown in Figure 3C and D, E64d increased the levels of LC3-II and SQSTM1 in both PK-15 and 3D4/2 cells, at 24 and 48 hpi, compared with control groups. Interestingly, E64d treatment also increased the level of the E2 protein during CSFV infection (Fig. 3C and D). These data indicate that a complete autophagic response can be induced in host cells following CSFV infection.

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