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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 2. Expression of autophagy marker proteins in CSFV-infected cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) for 6, 12, 24, 36, and 48 h. At the end of the infection, the expression of LC3, ATG12–ATG5, ATG5, BECN1, E2, and ACTB (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. (B) 3D4/2 cells were infected and analyzed as in (A). The relative levels of the targeted proteins were estimated by densitometry, 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.
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Figure 2: Figure 2. Expression of autophagy marker proteins in CSFV-infected cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) for 6, 12, 24, 36, and 48 h. At the end of the infection, the expression of LC3, ATG12–ATG5, ATG5, BECN1, E2, and ACTB (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. (B) 3D4/2 cells were infected and analyzed as in (A). The relative levels of the targeted proteins were estimated by densitometry, 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.

Mentions: To further analyze if the autophagy machinery can be triggered by CSFV infection, we examined the levels of autophagy marker proteins in CSFV-infected cells by using immunoblotting, including LC3 conversion, ATG12–ATG5 conjugation, and ATG5 and BECN1 expression. LC3 is widely used as a marker for assessing autophagy and correlates well with the formation of the autophagosome.32,33 Our results showed that LC3-II expression was upregulated in CSFV-infected PK-15 and 3D4/2 cells, with a decrease in LC3-I expression relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). We next examined the autophagy conjugation system by detecting the expression of ATG12–ATG5 and ATG5 by using a specific antibody to ATG5, which associates with the membranes of precursor autophagosomes.34,35 The CSFV-infected cells had an increased level of ATG12–ATG5 and ATG5 relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). We further detected the level of BECN1, which is involved in the early steps of the autophagy pathway.36 The results showed that CSFV infection generated the overexpression of BECN1 in host cells relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). Meanwhile, the detection of CSFV envelope protein E2 was used to estimate the progression of infection (Fig. 2A and B, lanes 6 to 10). More importantly, the level of autophagy marker proteins was near the detection limit in mock-infected cells (Fig. 2A and B, lanes 1 to 5), suggesting that a basal level of autophagy is present in normal PK-15 and 3D4/2 cells. In addition, the densitometric ratios of autophagy marker proteins (relative to ACTB) not only increased in CSFV-infected cells compared with uninfected (mock) cells, but also increased progressively with increase in infection time (Fig. 2A and B, lower part). These findings indicate that the early stages of autophagy are induced upon CSFV infection in host cells.


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 2. Expression of autophagy marker proteins in CSFV-infected cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) for 6, 12, 24, 36, and 48 h. At the end of the infection, the expression of LC3, ATG12–ATG5, ATG5, BECN1, E2, and ACTB (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. (B) 3D4/2 cells were infected and analyzed as in (A). The relative levels of the targeted proteins were estimated by densitometry, 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.
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Figure 2: Figure 2. Expression of autophagy marker proteins in CSFV-infected cells. (A) PK-15 cells were mock-infected or infected with CSFV (MOI = 1) or UV-inactivated CSFV (MOI = 1) for 6, 12, 24, 36, and 48 h. At the end of the infection, the expression of LC3, ATG12–ATG5, ATG5, BECN1, E2, and ACTB (loading control) were analyzed by immunoblotting with specific antibodies as described in Materials and Methods. (B) 3D4/2 cells were infected and analyzed as in (A). The relative levels of the targeted proteins were estimated by densitometry, 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.
Mentions: To further analyze if the autophagy machinery can be triggered by CSFV infection, we examined the levels of autophagy marker proteins in CSFV-infected cells by using immunoblotting, including LC3 conversion, ATG12–ATG5 conjugation, and ATG5 and BECN1 expression. LC3 is widely used as a marker for assessing autophagy and correlates well with the formation of the autophagosome.32,33 Our results showed that LC3-II expression was upregulated in CSFV-infected PK-15 and 3D4/2 cells, with a decrease in LC3-I expression relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). We next examined the autophagy conjugation system by detecting the expression of ATG12–ATG5 and ATG5 by using a specific antibody to ATG5, which associates with the membranes of precursor autophagosomes.34,35 The CSFV-infected cells had an increased level of ATG12–ATG5 and ATG5 relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). We further detected the level of BECN1, which is involved in the early steps of the autophagy pathway.36 The results showed that CSFV infection generated the overexpression of BECN1 in host cells relative to mock-infected cells (Fig. 2A and B, lanes 1 to 10). Meanwhile, the detection of CSFV envelope protein E2 was used to estimate the progression of infection (Fig. 2A and B, lanes 6 to 10). More importantly, the level of autophagy marker proteins was near the detection limit in mock-infected cells (Fig. 2A and B, lanes 1 to 5), suggesting that a basal level of autophagy is present in normal PK-15 and 3D4/2 cells. In addition, the densitometric ratios of autophagy marker proteins (relative to ACTB) not only increased in CSFV-infected cells compared with uninfected (mock) cells, but also increased progressively with increase in infection time (Fig. 2A and B, lower part). These findings indicate that the early stages of autophagy are induced upon CSFV infection in host cells.

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