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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

Measurement of the autophagic flux in HeLa cells infected with CA16.(A, B) Western blotting of cells with autophagy inhibited by CQ. HeLa cells were pretreated with CQ for 4 h, followed by infection with CA16 at an MOI of 2. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium in the absence or presence of CQ. At 24 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B and Vp1 antibodies. (C) Western blotting analysis of p62 protein expression in HeLa cells infected with CA16. Cells were infected with CA16 at an MOI of 1. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium. Cells were harvested at the indicated time points and detected with anti-p62 antibody compared to uninfected control cells. (D) HeLa cells transfected with ptfLC3 were infected with CA16 (MOI = 1) or treated with CQ or HBSS. The cells were collected, fixed, and visualized at 24 h postinfection. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. Scale bar, 10μm. (E) Western blotting of autophagy-related proteins in cells transfected with the indicated shRNA and determination of CA16 replication in these transfected cells. HeLa cells were transfected with either specific shRNA targeting Beclin 1, Atg5 or scrambled shRNA. At 48 h after transfection, cells were infected with CA16 at an MOI of 2. Samples were collected at 12 h after infection with CA16 and detected with anti-Beclin 1,-Atg5 and-Vp1 antibodies. β-Actin was used as a protein loading control. The extracellular virus yields were determined at 48 hpi and expressed as lgTCID50/ml. 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, ***P< 0.001. (F) Determination of CA16 replication in HeLa cells treated with CQ. HeLa cells were pretreated with CQ for 4 h followed by infection with CA16 at an MOI of 2. The extracellular virus yields were determined at 12 hpi and expressed as lgTCID50/ml. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05.
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pone.0122109.g002: Measurement of the autophagic flux in HeLa cells infected with CA16.(A, B) Western blotting of cells with autophagy inhibited by CQ. HeLa cells were pretreated with CQ for 4 h, followed by infection with CA16 at an MOI of 2. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium in the absence or presence of CQ. At 24 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B and Vp1 antibodies. (C) Western blotting analysis of p62 protein expression in HeLa cells infected with CA16. Cells were infected with CA16 at an MOI of 1. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium. Cells were harvested at the indicated time points and detected with anti-p62 antibody compared to uninfected control cells. (D) HeLa cells transfected with ptfLC3 were infected with CA16 (MOI = 1) or treated with CQ or HBSS. The cells were collected, fixed, and visualized at 24 h postinfection. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. Scale bar, 10μm. (E) Western blotting of autophagy-related proteins in cells transfected with the indicated shRNA and determination of CA16 replication in these transfected cells. HeLa cells were transfected with either specific shRNA targeting Beclin 1, Atg5 or scrambled shRNA. At 48 h after transfection, cells were infected with CA16 at an MOI of 2. Samples were collected at 12 h after infection with CA16 and detected with anti-Beclin 1,-Atg5 and-Vp1 antibodies. β-Actin was used as a protein loading control. The extracellular virus yields were determined at 48 hpi and expressed as lgTCID50/ml. 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, ***P< 0.001. (F) Determination of CA16 replication in HeLa cells treated with CQ. HeLa cells were pretreated with CQ for 4 h followed by infection with CA16 at an MOI of 2. The extracellular virus yields were determined at 12 hpi and expressed as lgTCID50/ml. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05.

Mentions: Because autophagosomes are merely the intermediate products of the autophagy process, the accumulation of autophagosomes may be the result of either increased autophagosome biogenesis or disrupted trafficking to lysosomes for degradation. In other words, autophagosome accumulation may result from autophagy induction (completed autophagy or incomplete autophagy) or impaired autophagosome-lysosome fusion[9,10]. To clarify which of these mechanisms was responsible for the increased numbers of autophagosomes, we employed CQ (a widely used compound that prevents autophagosome-lysosome fusion by elevating the pH of the lysosomes)[10]. As shown in Fig 2A, higher levels of LC3-II accumulated in CA16-infected cells compared to mock-infected cells (Fig 2A, lanes 2 and 3) following CQ treatment, indicating that the accumulation of autophagosomes was not caused by blocking basic autophagy but by new autophagosome formation. However, no differences in LC3-II levels were observed between CA16-infected cells (moi = 2) treated with CQ compared to the untreated controls (Fig 2B, lanes 2 and 3), indicating that CA16 infection may play a similar role to CQ in blocking autophagosome maturation. Additionally, we also analyzed p62 protein levels in CA16-infected cells. P62 is another widely used autophagy flux marker because p62 links both LC3 and the ubiquitinated substrates that are degraded in the completed autophagy process after the autophagosomes fuse with lysosomes[9,10]. We failed to observe obvious degradation of p62 in CA16-infected HeLa cells even 24 hr after infection, although nearly half of the cells were severely cytopathic (S2B Fig). Inversely, higher levels of p62 were shown with the progression of CA16 infection (Fig 2C).


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)

Measurement of the autophagic flux in HeLa cells infected with CA16.(A, B) Western blotting of cells with autophagy inhibited by CQ. HeLa cells were pretreated with CQ for 4 h, followed by infection with CA16 at an MOI of 2. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium in the absence or presence of CQ. At 24 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B and Vp1 antibodies. (C) Western blotting analysis of p62 protein expression in HeLa cells infected with CA16. Cells were infected with CA16 at an MOI of 1. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium. Cells were harvested at the indicated time points and detected with anti-p62 antibody compared to uninfected control cells. (D) HeLa cells transfected with ptfLC3 were infected with CA16 (MOI = 1) or treated with CQ or HBSS. The cells were collected, fixed, and visualized at 24 h postinfection. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. Scale bar, 10μm. (E) Western blotting of autophagy-related proteins in cells transfected with the indicated shRNA and determination of CA16 replication in these transfected cells. HeLa cells were transfected with either specific shRNA targeting Beclin 1, Atg5 or scrambled shRNA. At 48 h after transfection, cells were infected with CA16 at an MOI of 2. Samples were collected at 12 h after infection with CA16 and detected with anti-Beclin 1,-Atg5 and-Vp1 antibodies. β-Actin was used as a protein loading control. The extracellular virus yields were determined at 48 hpi and expressed as lgTCID50/ml. 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, ***P< 0.001. (F) Determination of CA16 replication in HeLa cells treated with CQ. HeLa cells were pretreated with CQ for 4 h followed by infection with CA16 at an MOI of 2. The extracellular virus yields were determined at 12 hpi and expressed as lgTCID50/ml. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05.
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pone.0122109.g002: Measurement of the autophagic flux in HeLa cells infected with CA16.(A, B) Western blotting of cells with autophagy inhibited by CQ. HeLa cells were pretreated with CQ for 4 h, followed by infection with CA16 at an MOI of 2. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium in the absence or presence of CQ. At 24 h after infection with CA16, the cells were subjected to Western blotting using anti-LC3B and Vp1 antibodies. (C) Western blotting analysis of p62 protein expression in HeLa cells infected with CA16. Cells were infected with CA16 at an MOI of 1. After 1 h of virus absorption at 37°C, the cells were further cultured in maintenance medium. Cells were harvested at the indicated time points and detected with anti-p62 antibody compared to uninfected control cells. (D) HeLa cells transfected with ptfLC3 were infected with CA16 (MOI = 1) or treated with CQ or HBSS. The cells were collected, fixed, and visualized at 24 h postinfection. The graph shows the quantification of autophagosomes by calculating the average number of dots in 20 cells. Scale bar, 10μm. (E) Western blotting of autophagy-related proteins in cells transfected with the indicated shRNA and determination of CA16 replication in these transfected cells. HeLa cells were transfected with either specific shRNA targeting Beclin 1, Atg5 or scrambled shRNA. At 48 h after transfection, cells were infected with CA16 at an MOI of 2. Samples were collected at 12 h after infection with CA16 and detected with anti-Beclin 1,-Atg5 and-Vp1 antibodies. β-Actin was used as a protein loading control. The extracellular virus yields were determined at 48 hpi and expressed as lgTCID50/ml. 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, ***P< 0.001. (F) Determination of CA16 replication in HeLa cells treated with CQ. HeLa cells were pretreated with CQ for 4 h followed by infection with CA16 at an MOI of 2. The extracellular virus yields were determined at 12 hpi and expressed as lgTCID50/ml. Data are presented as the means from three independent experiments. Significance was analyzed with a two-tailed Student’s t test. *P< 0.05.
Mentions: Because autophagosomes are merely the intermediate products of the autophagy process, the accumulation of autophagosomes may be the result of either increased autophagosome biogenesis or disrupted trafficking to lysosomes for degradation. In other words, autophagosome accumulation may result from autophagy induction (completed autophagy or incomplete autophagy) or impaired autophagosome-lysosome fusion[9,10]. To clarify which of these mechanisms was responsible for the increased numbers of autophagosomes, we employed CQ (a widely used compound that prevents autophagosome-lysosome fusion by elevating the pH of the lysosomes)[10]. As shown in Fig 2A, higher levels of LC3-II accumulated in CA16-infected cells compared to mock-infected cells (Fig 2A, lanes 2 and 3) following CQ treatment, indicating that the accumulation of autophagosomes was not caused by blocking basic autophagy but by new autophagosome formation. However, no differences in LC3-II levels were observed between CA16-infected cells (moi = 2) treated with CQ compared to the untreated controls (Fig 2B, lanes 2 and 3), indicating that CA16 infection may play a similar role to CQ in blocking autophagosome maturation. Additionally, we also analyzed p62 protein levels in CA16-infected cells. P62 is another widely used autophagy flux marker because p62 links both LC3 and the ubiquitinated substrates that are degraded in the completed autophagy process after the autophagosomes fuse with lysosomes[9,10]. We failed to observe obvious degradation of p62 in CA16-infected HeLa cells even 24 hr after infection, although nearly half of the cells were severely cytopathic (S2B Fig). Inversely, higher levels of p62 were shown with the progression of CA16 infection (Fig 2C).

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