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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 11. Pharmacological alteration of autophagy does not affect cell viability. The cell viability of PK-15 (A) and 3D4/2 (B) cells were determined by the MTT assay after treatments with rapamycin (100 nM) or 3-MA (5 mM) for 48 h. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; #P > 0.05.
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Figure 11: Figure 11. Pharmacological alteration of autophagy does not affect cell viability. The cell viability of PK-15 (A) and 3D4/2 (B) cells were determined by the MTT assay after treatments with rapamycin (100 nM) or 3-MA (5 mM) for 48 h. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; #P > 0.05.

Mentions: To determine whether the pharmacological alteration of autophagy with rapamycin and 3-MA affected the capability of CSFV replication by changing the cell viability, we performed the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to analyze the effects of these autophagic reagents on cell viability. Statistical analyses revealed no significant effects on the viability of cells treated with rapamycin or 3-MA (P > 0.05) (Fig. 11).


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 11. Pharmacological alteration of autophagy does not affect cell viability. The cell viability of PK-15 (A) and 3D4/2 (B) cells were determined by the MTT assay after treatments with rapamycin (100 nM) or 3-MA (5 mM) for 48 h. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; #P > 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389882&req=5

Figure 11: Figure 11. Pharmacological alteration of autophagy does not affect cell viability. The cell viability of PK-15 (A) and 3D4/2 (B) cells were determined by the MTT assay after treatments with rapamycin (100 nM) or 3-MA (5 mM) for 48 h. The data represent the mean ± SD of 3 independent experiments. Two-way ANOVA; #P > 0.05.
Mentions: To determine whether the pharmacological alteration of autophagy with rapamycin and 3-MA affected the capability of CSFV replication by changing the cell viability, we performed the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to analyze the effects of these autophagic reagents on cell viability. Statistical analyses revealed no significant effects on the viability of cells treated with rapamycin or 3-MA (P > 0.05) (Fig. 11).

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