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Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Shi Y, He X, Zhu G, Tu H, Liu Z, Li W, Han S, Yin J, Peng B, Liu W - PLoS ONE (2015)

Bottom Line: Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy.In summary, CA16 can use autophagy to enhance its own replication.These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Organism and Infectious Diseases Research Institute, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071, China; Hubei Province Key Laboratory of Allergy and Immunology, Wuhan, 430071, China.

ABSTRACT
Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

No MeSH data available.


Related in: MedlinePlus

Expression of CA16 2C and 3C protein increase autophagosome accumulation.(A) Genome structure of CA16. (B) WB analysis of LC3 protein expression in HeLa cells transfected with plasmids expressing individual viral proteins. Cells transfected with the pCMV-HA empty vector or plasmids expressing CA16 non-structural proteins 2A, 2B, 2C, 3AB, 3C or 3D. Cells were harvested at 24 h after transfection, and protein expression was detected with anti-LC3B and HA antibodies. Rapamycin-treated cells were used as a positive control, and β-Actin was used as a protein loading control. Representative results are shown with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01. (C) GFP-LC3 aggregation was visualized via fluorescence microscopy. HeLa cells were transfected with vector or HA-2C/3C plus pEGFP-LC3 for 24 h, and the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown. The number of GFP-LC3 dots in each cell was counted, and the graph shows the quantification of autophagosomes by taking the average number of dots in 20 cells. Scale bar, 10μm. (D) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells transfected with vector or viral proteins were processed and analyzed at 24 h after transfection for the accumulation of autophagosomes via electron microscopy. White arrows indicate representative autophagosomes. (d, f) represent the higher-magnification views of (c, e).
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pone.0122109.g003: Expression of CA16 2C and 3C protein increase autophagosome accumulation.(A) Genome structure of CA16. (B) WB analysis of LC3 protein expression in HeLa cells transfected with plasmids expressing individual viral proteins. Cells transfected with the pCMV-HA empty vector or plasmids expressing CA16 non-structural proteins 2A, 2B, 2C, 3AB, 3C or 3D. Cells were harvested at 24 h after transfection, and protein expression was detected with anti-LC3B and HA antibodies. Rapamycin-treated cells were used as a positive control, and β-Actin was used as a protein loading control. Representative results are shown with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01. (C) GFP-LC3 aggregation was visualized via fluorescence microscopy. HeLa cells were transfected with vector or HA-2C/3C plus pEGFP-LC3 for 24 h, and the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown. The number of GFP-LC3 dots in each cell was counted, and the graph shows the quantification of autophagosomes by taking the average number of dots in 20 cells. Scale bar, 10μm. (D) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells transfected with vector or viral proteins were processed and analyzed at 24 h after transfection for the accumulation of autophagosomes via electron microscopy. White arrows indicate representative autophagosomes. (d, f) represent the higher-magnification views of (c, e).

Mentions: The results obtained here imply that CA16 infection resulted in autophagosome accumulation in infected HeLa cells. However, it was unclear whether viral proteins played a role in activating autophagy in CA16-infected cells. To address this question, HeLa cells were transfected with plasmids over-expressing each viral protein (2A, 2B, 2C, 3AB, 3C, and 3D, Fig 3A) of CA16. As presented in Fig 3B, scanning analysis revealed that the LC3-II/Actin ratios were higher compared to the controls in HeLa cells over-expressing the 2C (P< 0.01), 3C (P< 0.05), and 3D (P< 0.05) proteins but not the 2A, 2B, or 3AB proteins. Similar results were demonstrated in RD cells over-expressing the 2C (P< 0.001) and 3C (P< 0.05) proteins but not the 3D protein (S1B Fig). Thus, the 2C and 3C proteins with the capacity to trigger autophagosome formation in both HeLa and RD cells were chosen for further study.


Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Shi Y, He X, Zhu G, Tu H, Liu Z, Li W, Han S, Yin J, Peng B, Liu W - PLoS ONE (2015)

Expression of CA16 2C and 3C protein increase autophagosome accumulation.(A) Genome structure of CA16. (B) WB analysis of LC3 protein expression in HeLa cells transfected with plasmids expressing individual viral proteins. Cells transfected with the pCMV-HA empty vector or plasmids expressing CA16 non-structural proteins 2A, 2B, 2C, 3AB, 3C or 3D. Cells were harvested at 24 h after transfection, and protein expression was detected with anti-LC3B and HA antibodies. Rapamycin-treated cells were used as a positive control, and β-Actin was used as a protein loading control. Representative results are shown with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01. (C) GFP-LC3 aggregation was visualized via fluorescence microscopy. HeLa cells were transfected with vector or HA-2C/3C plus pEGFP-LC3 for 24 h, and the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown. The number of GFP-LC3 dots in each cell was counted, and the graph shows the quantification of autophagosomes by taking the average number of dots in 20 cells. Scale bar, 10μm. (D) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells transfected with vector or viral proteins were processed and analyzed at 24 h after transfection for the accumulation of autophagosomes via electron microscopy. White arrows indicate representative autophagosomes. (d, f) represent the higher-magnification views of (c, e).
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pone.0122109.g003: Expression of CA16 2C and 3C protein increase autophagosome accumulation.(A) Genome structure of CA16. (B) WB analysis of LC3 protein expression in HeLa cells transfected with plasmids expressing individual viral proteins. Cells transfected with the pCMV-HA empty vector or plasmids expressing CA16 non-structural proteins 2A, 2B, 2C, 3AB, 3C or 3D. Cells were harvested at 24 h after transfection, and protein expression was detected with anti-LC3B and HA antibodies. Rapamycin-treated cells were used as a positive control, and β-Actin was used as a protein loading control. Representative results are shown with graphs representing the ratio of LC3-II to β-Actin normalized to the control condition. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05, **P< 0.01. (C) GFP-LC3 aggregation was visualized via fluorescence microscopy. HeLa cells were transfected with vector or HA-2C/3C plus pEGFP-LC3 for 24 h, and the GFP-LC3 aggregations in the cells were assessed via fluorescence microscopy. Representative images are shown. The number of GFP-LC3 dots in each cell was counted, and the graph shows the quantification of autophagosomes by taking the average number of dots in 20 cells. Scale bar, 10μm. (D) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells transfected with vector or viral proteins were processed and analyzed at 24 h after transfection for the accumulation of autophagosomes via electron microscopy. White arrows indicate representative autophagosomes. (d, f) represent the higher-magnification views of (c, e).
Mentions: The results obtained here imply that CA16 infection resulted in autophagosome accumulation in infected HeLa cells. However, it was unclear whether viral proteins played a role in activating autophagy in CA16-infected cells. To address this question, HeLa cells were transfected with plasmids over-expressing each viral protein (2A, 2B, 2C, 3AB, 3C, and 3D, Fig 3A) of CA16. As presented in Fig 3B, scanning analysis revealed that the LC3-II/Actin ratios were higher compared to the controls in HeLa cells over-expressing the 2C (P< 0.01), 3C (P< 0.05), and 3D (P< 0.05) proteins but not the 2A, 2B, or 3AB proteins. Similar results were demonstrated in RD cells over-expressing the 2C (P< 0.001) and 3C (P< 0.05) proteins but not the 3D protein (S1B Fig). Thus, the 2C and 3C proteins with the capacity to trigger autophagosome formation in both HeLa and RD cells were chosen for further study.

Bottom Line: Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy.In summary, CA16 can use autophagy to enhance its own replication.These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

View Article: PubMed Central - PubMed

Affiliation: Pathogenic Organism and Infectious Diseases Research Institute, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071, China; Hubei Province Key Laboratory of Allergy and Immunology, Wuhan, 430071, China.

ABSTRACT
Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

No MeSH data available.


Related in: MedlinePlus