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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 7. Induction of autophagy with rapamycin enhances CSFV replication. (A and D) PK-15 (A) and 3D4/2 (D) cells were pretreated with rapamycin (100 nM) or DMSO (Control) for 1 h, followed by CSFV adsorption for 1 h at an MOI of 0.5. The cells were further cultured in fresh medium in the absence or presence of rapamycin (100 nM). At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against LC3B, CSFV-E2, and ACTB (loading control). The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01. (B and E) PK-15 (B) and 3D4/2 (E) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, 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 < 0.05; **P < 0.01; ***P < 0.001. (C and F) PK-15 (C) and 3D4/2 (F) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using an immunofluorescence assay as 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 7: Figure 7. Induction of autophagy with rapamycin enhances CSFV replication. (A and D) PK-15 (A) and 3D4/2 (D) cells were pretreated with rapamycin (100 nM) or DMSO (Control) for 1 h, followed by CSFV adsorption for 1 h at an MOI of 0.5. The cells were further cultured in fresh medium in the absence or presence of rapamycin (100 nM). At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against LC3B, CSFV-E2, and ACTB (loading control). The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01. (B and E) PK-15 (B) and 3D4/2 (E) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, 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 < 0.05; **P < 0.01; ***P < 0.001. (C and F) PK-15 (C) and 3D4/2 (F) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using an immunofluorescence assay as 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: Rapamycin, an inducer of autophagy, can activate autophagy by blocking the mechanistic target of rapamycin (MTOR) pathway.42-44 To analyze the role of autophagy in the replication of CSFV, we examined viral envelope protein E2 expression and viral progeny yields following rapamycin treatment. We found that the induction of autophagy with rapamycin not only upregulated the expression of LC3-II and CSFV-E2 at 24 and 48 hpi in CSFV-infected PK-15 cells (Fig. 7A), but also increased the yields of CSFV progeny (Fig. 7B and C). Notably, the effect of rapamycin treatment on the extracellular viral load and titer was greater than the intracellular load and titer (Fig. 7B and C). Similar results for viral envelope protein E2 expression and viral progeny yields were also obtained in treated 3D4/2 cells (Fig. 7D–F). These data suggest that the autophagy mechanism may support 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 7. Induction of autophagy with rapamycin enhances CSFV replication. (A and D) PK-15 (A) and 3D4/2 (D) cells were pretreated with rapamycin (100 nM) or DMSO (Control) for 1 h, followed by CSFV adsorption for 1 h at an MOI of 0.5. The cells were further cultured in fresh medium in the absence or presence of rapamycin (100 nM). At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against LC3B, CSFV-E2, and ACTB (loading control). The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01. (B and E) PK-15 (B) and 3D4/2 (E) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, 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 < 0.05; **P < 0.01; ***P < 0.001. (C and F) PK-15 (C) and 3D4/2 (F) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using an immunofluorescence assay as 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 7: Figure 7. Induction of autophagy with rapamycin enhances CSFV replication. (A and D) PK-15 (A) and 3D4/2 (D) cells were pretreated with rapamycin (100 nM) or DMSO (Control) for 1 h, followed by CSFV adsorption for 1 h at an MOI of 0.5. The cells were further cultured in fresh medium in the absence or presence of rapamycin (100 nM). At 24 and 48 hpi, cell samples were analyzed by immunoblotting with antibodies against LC3B, CSFV-E2, and ACTB (loading control). The relative levels of the targeted proteins were estimated by densitometric scanning, and the ratios were calculated relative to ACTB. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; *P < 0.05; **P < 0.01. (B and E) PK-15 (B) and 3D4/2 (E) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, 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 < 0.05; **P < 0.01; ***P < 0.001. (C and F) PK-15 (C) and 3D4/2 (F) cells were pretreated and infected as described in (A and D). At 24 and 48 hpi, both the extracellular and intracellular virus titers were measured by endpoint dilution titrations by using an immunofluorescence assay as 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: Rapamycin, an inducer of autophagy, can activate autophagy by blocking the mechanistic target of rapamycin (MTOR) pathway.42-44 To analyze the role of autophagy in the replication of CSFV, we examined viral envelope protein E2 expression and viral progeny yields following rapamycin treatment. We found that the induction of autophagy with rapamycin not only upregulated the expression of LC3-II and CSFV-E2 at 24 and 48 hpi in CSFV-infected PK-15 cells (Fig. 7A), but also increased the yields of CSFV progeny (Fig. 7B and C). Notably, the effect of rapamycin treatment on the extracellular viral load and titer was greater than the intracellular load and titer (Fig. 7B and C). Similar results for viral envelope protein E2 expression and viral progeny yields were also obtained in treated 3D4/2 cells (Fig. 7D–F). These data suggest that the autophagy mechanism may support 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