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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

CA16 infection triggers autophagy in infected cells.(A) Western blotting (WB) analysis of LC3 protein expression in HeLa cells infected with Coxsackievirus A16 (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 blotted with anti-LC3B and Vp1 antibodies; the results were compared to uninfected control cells. (B) GFP-LC3 dots and viral proteins expression were visualized via confocal microscopy. HeLa cells were transfected with GFP-LC3 plasmid for 24 h, followed by CA16 infection(MOI = 1) or treated with Rapamycin and the GFP-LC3 aggregations in the cells were assessed via confocal microscopy. The localization of virus was determined by indirect immunofluorescence with a human anti-CA16 sera and TRITC-conjugated anti-human IgG. Representative images are shown. Scale bar, 8μm. (C) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells infected with CA16 (MOI = 1) were processed and analyzed at 24 hpi for the accumulation of autophagosome via electron microscopy. White arrows indicate representative autophagosomes. (c) represents the higher-magnification views of (b).
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pone.0122109.g001: CA16 infection triggers autophagy in infected cells.(A) Western blotting (WB) analysis of LC3 protein expression in HeLa cells infected with Coxsackievirus A16 (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 blotted with anti-LC3B and Vp1 antibodies; the results were compared to uninfected control cells. (B) GFP-LC3 dots and viral proteins expression were visualized via confocal microscopy. HeLa cells were transfected with GFP-LC3 plasmid for 24 h, followed by CA16 infection(MOI = 1) or treated with Rapamycin and the GFP-LC3 aggregations in the cells were assessed via confocal microscopy. The localization of virus was determined by indirect immunofluorescence with a human anti-CA16 sera and TRITC-conjugated anti-human IgG. Representative images are shown. Scale bar, 8μm. (C) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells infected with CA16 (MOI = 1) were processed and analyzed at 24 hpi for the accumulation of autophagosome via electron microscopy. White arrows indicate representative autophagosomes. (c) represents the higher-magnification views of (b).

Mentions: To illuminate whether CA16 could induce autophagy in infected cells, we first performed Western blotting analysis to detect the expression of LC3, which is a hallmark of autophagy. Because the LC3 precursor (LC3-I) is diffusely localized in the cytosol in resting state cells and can be quickly converted to the lipidated, autophagosome-associated form (LC3-II) after autophagy induction, the conversion of LC3-I to LC3-II is widely used to evaluate autophagic activity[9,10]. As shown in Fig 1A, the intensity of the LC3-II band was increased in CA16-infected cells relative to mock-infected cells as infection progressed. At the same time, a human polyclonal anti-CA16 serum was applied to track the progression of CA16 infection. These results showed that LC3-II levels correlated well with VP1 protein expression, indicating that the autophagy was indeed induced by CA16 infection (Fig 1A). LC3-II levels were increased in cells treated with rapamycin, a commonly used autophagy inducer. Likewise, a marked turnover of LC3-I to LC3-II was also observed in CA16-infected RD cells (S1A Fig).


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)

CA16 infection triggers autophagy in infected cells.(A) Western blotting (WB) analysis of LC3 protein expression in HeLa cells infected with Coxsackievirus A16 (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 blotted with anti-LC3B and Vp1 antibodies; the results were compared to uninfected control cells. (B) GFP-LC3 dots and viral proteins expression were visualized via confocal microscopy. HeLa cells were transfected with GFP-LC3 plasmid for 24 h, followed by CA16 infection(MOI = 1) or treated with Rapamycin and the GFP-LC3 aggregations in the cells were assessed via confocal microscopy. The localization of virus was determined by indirect immunofluorescence with a human anti-CA16 sera and TRITC-conjugated anti-human IgG. Representative images are shown. Scale bar, 8μm. (C) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells infected with CA16 (MOI = 1) were processed and analyzed at 24 hpi for the accumulation of autophagosome via electron microscopy. White arrows indicate representative autophagosomes. (c) represents the higher-magnification views of (b).
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pone.0122109.g001: CA16 infection triggers autophagy in infected cells.(A) Western blotting (WB) analysis of LC3 protein expression in HeLa cells infected with Coxsackievirus A16 (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 blotted with anti-LC3B and Vp1 antibodies; the results were compared to uninfected control cells. (B) GFP-LC3 dots and viral proteins expression were visualized via confocal microscopy. HeLa cells were transfected with GFP-LC3 plasmid for 24 h, followed by CA16 infection(MOI = 1) or treated with Rapamycin and the GFP-LC3 aggregations in the cells were assessed via confocal microscopy. The localization of virus was determined by indirect immunofluorescence with a human anti-CA16 sera and TRITC-conjugated anti-human IgG. Representative images are shown. Scale bar, 8μm. (C) Autophagic vacuoles were detected via transmission electron microscopy (TEM). HeLa cells infected with CA16 (MOI = 1) were processed and analyzed at 24 hpi for the accumulation of autophagosome via electron microscopy. White arrows indicate representative autophagosomes. (c) represents the higher-magnification views of (b).
Mentions: To illuminate whether CA16 could induce autophagy in infected cells, we first performed Western blotting analysis to detect the expression of LC3, which is a hallmark of autophagy. Because the LC3 precursor (LC3-I) is diffusely localized in the cytosol in resting state cells and can be quickly converted to the lipidated, autophagosome-associated form (LC3-II) after autophagy induction, the conversion of LC3-I to LC3-II is widely used to evaluate autophagic activity[9,10]. As shown in Fig 1A, the intensity of the LC3-II band was increased in CA16-infected cells relative to mock-infected cells as infection progressed. At the same time, a human polyclonal anti-CA16 serum was applied to track the progression of CA16 infection. These results showed that LC3-II levels correlated well with VP1 protein expression, indicating that the autophagy was indeed induced by CA16 infection (Fig 1A). LC3-II levels were increased in cells treated with rapamycin, a commonly used autophagy inducer. Likewise, a marked turnover of LC3-I to LC3-II was also observed in CA16-infected RD cells (S1A Fig).

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