Limits...
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

IRGM interacts with Atg5 and Atg10 and the IRGM promoter can be activated by 2C.(A) LC3-I/LC3-II and IRGM expression in HeLa cells infected with Coxsackievirus A16 (CA16). Cells were infected with CA16 at an MOI of 2, harvested at 24 hpi. Detection with anti-IRGM, LC3B and Vp1 antibodies was compared to control uninfected cells. (B) Real time-PCR analysis of IRGM mRNA levels in Hela cells that infected or not with CA16 (n = 3, ***P<0.001). (C) Endogenous IRGM interacts with endogenous Atg5 and Atg10 in CA16 infected cells. HeLa cells were infected with CA16 (MOI = 1) for 24h. Whole-cell lysates (WCL) were subjected to coimmunoprecipitation (Co-IP) with IRGM antibody or IgG, followed by SDS-PAGE/immunoblot analysis with antibodies as indicated. (D) Effects of 2C and 3C on IRGM promoter activation. HeLa cells were co-transfected with HA-2C or HA-3C and IRGM-Luc. Plasmids expressing HA and pRL-TK were used as controls. At 24 h after transfection, cells lysates were assayed for luciferase activity. Data are representative of three independent experiments with triplicate samples. **P< 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4390341&req=5

pone.0122109.g005: IRGM interacts with Atg5 and Atg10 and the IRGM promoter can be activated by 2C.(A) LC3-I/LC3-II and IRGM expression in HeLa cells infected with Coxsackievirus A16 (CA16). Cells were infected with CA16 at an MOI of 2, harvested at 24 hpi. Detection with anti-IRGM, LC3B and Vp1 antibodies was compared to control uninfected cells. (B) Real time-PCR analysis of IRGM mRNA levels in Hela cells that infected or not with CA16 (n = 3, ***P<0.001). (C) Endogenous IRGM interacts with endogenous Atg5 and Atg10 in CA16 infected cells. HeLa cells were infected with CA16 (MOI = 1) for 24h. Whole-cell lysates (WCL) were subjected to coimmunoprecipitation (Co-IP) with IRGM antibody or IgG, followed by SDS-PAGE/immunoblot analysis with antibodies as indicated. (D) Effects of 2C and 3C on IRGM promoter activation. HeLa cells were co-transfected with HA-2C or HA-3C and IRGM-Luc. Plasmids expressing HA and pRL-TK were used as controls. At 24 h after transfection, cells lysates were assayed for luciferase activity. Data are representative of three independent experiments with triplicate samples. **P< 0.01.

Mentions: Recently, the human immunity-related GTPase family M protein (IRGM, also known as interferon-inducible protein 1 (IFI1)) was reported to be widely targeted by several RNA viruses that are capable of inducing autophagy in human cells to facilitate viral replication. However, whether and how IRGM regulates autophagy upon CA16 infection is unknown[20,21,22]. To better understand the molecular mechanism that mediates the autophagy process, we explored the relationship between IRGM expression and autophagy induction. As shown in Fig 5A and 5B, Western blotting and real-time PCR were performed and we found that CA16 infection triggered significant increases in both the mRNA (Fig 5B, P< 0.001) and protein (Fig 5A, P< 0.001) levels of IRGM. The LC3-I-to-LC3-II conversion was also notably enhanced following CA16 infection (P< 0.01). Thus, the LC3-II level positively correlated with IRGM expression (Fig 5A). Because IRGM levels increased with autophagy induction, the findings led us to question whether IRGM was able to regulate the autophagic process by interacting with certain human autophagy proteins. As confirmed by coimmunoprecipitation (Co-IP), endogenous IRGM colocalized with endogenous Atg5 (Fig 5C). Furthermore, a physical interaction between IRGM and Atg10 was also confirmed (Fig 5C). In addition, we also have assessed the exogenous Atg5-IRGM and exogenous Atg10-IRGM interaction upon CA16 infection. Similar effects were also observed (S3 Fig). Next, to evaluate whether over-expression of viral protein 2C or 3C had an effect on IRGM promoter activity, HeLa cells were cotransfected with viral protein 2C or 3C and IRGM-promoter-luc. Luciferase activity was measured 24 h after transfection. The data showed that the relative luciferase activities of the IRGM promoter were markedly promoted in cells over-expressing 2C (P< 0.01), but not in cells over-expressing 3C (Fig 5D). Additionally, we performed a Co-IP assay to detect the physical interaction between IRGM and 2C or 3C (S3C Fig). Regrettably, we failed to confirm the direct interaction between the viral proteins and IRGM. Together, our results suggested that it is possible that 2C could enhance IRGM promoter activation, resulting in increased IRGM expression and the induction of autophagy.


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)

IRGM interacts with Atg5 and Atg10 and the IRGM promoter can be activated by 2C.(A) LC3-I/LC3-II and IRGM expression in HeLa cells infected with Coxsackievirus A16 (CA16). Cells were infected with CA16 at an MOI of 2, harvested at 24 hpi. Detection with anti-IRGM, LC3B and Vp1 antibodies was compared to control uninfected cells. (B) Real time-PCR analysis of IRGM mRNA levels in Hela cells that infected or not with CA16 (n = 3, ***P<0.001). (C) Endogenous IRGM interacts with endogenous Atg5 and Atg10 in CA16 infected cells. HeLa cells were infected with CA16 (MOI = 1) for 24h. Whole-cell lysates (WCL) were subjected to coimmunoprecipitation (Co-IP) with IRGM antibody or IgG, followed by SDS-PAGE/immunoblot analysis with antibodies as indicated. (D) Effects of 2C and 3C on IRGM promoter activation. HeLa cells were co-transfected with HA-2C or HA-3C and IRGM-Luc. Plasmids expressing HA and pRL-TK were used as controls. At 24 h after transfection, cells lysates were assayed for luciferase activity. Data are representative of three independent experiments with triplicate samples. **P< 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390341&req=5

pone.0122109.g005: IRGM interacts with Atg5 and Atg10 and the IRGM promoter can be activated by 2C.(A) LC3-I/LC3-II and IRGM expression in HeLa cells infected with Coxsackievirus A16 (CA16). Cells were infected with CA16 at an MOI of 2, harvested at 24 hpi. Detection with anti-IRGM, LC3B and Vp1 antibodies was compared to control uninfected cells. (B) Real time-PCR analysis of IRGM mRNA levels in Hela cells that infected or not with CA16 (n = 3, ***P<0.001). (C) Endogenous IRGM interacts with endogenous Atg5 and Atg10 in CA16 infected cells. HeLa cells were infected with CA16 (MOI = 1) for 24h. Whole-cell lysates (WCL) were subjected to coimmunoprecipitation (Co-IP) with IRGM antibody or IgG, followed by SDS-PAGE/immunoblot analysis with antibodies as indicated. (D) Effects of 2C and 3C on IRGM promoter activation. HeLa cells were co-transfected with HA-2C or HA-3C and IRGM-Luc. Plasmids expressing HA and pRL-TK were used as controls. At 24 h after transfection, cells lysates were assayed for luciferase activity. Data are representative of three independent experiments with triplicate samples. **P< 0.01.
Mentions: Recently, the human immunity-related GTPase family M protein (IRGM, also known as interferon-inducible protein 1 (IFI1)) was reported to be widely targeted by several RNA viruses that are capable of inducing autophagy in human cells to facilitate viral replication. However, whether and how IRGM regulates autophagy upon CA16 infection is unknown[20,21,22]. To better understand the molecular mechanism that mediates the autophagy process, we explored the relationship between IRGM expression and autophagy induction. As shown in Fig 5A and 5B, Western blotting and real-time PCR were performed and we found that CA16 infection triggered significant increases in both the mRNA (Fig 5B, P< 0.001) and protein (Fig 5A, P< 0.001) levels of IRGM. The LC3-I-to-LC3-II conversion was also notably enhanced following CA16 infection (P< 0.01). Thus, the LC3-II level positively correlated with IRGM expression (Fig 5A). Because IRGM levels increased with autophagy induction, the findings led us to question whether IRGM was able to regulate the autophagic process by interacting with certain human autophagy proteins. As confirmed by coimmunoprecipitation (Co-IP), endogenous IRGM colocalized with endogenous Atg5 (Fig 5C). Furthermore, a physical interaction between IRGM and Atg10 was also confirmed (Fig 5C). In addition, we also have assessed the exogenous Atg5-IRGM and exogenous Atg10-IRGM interaction upon CA16 infection. Similar effects were also observed (S3 Fig). Next, to evaluate whether over-expression of viral protein 2C or 3C had an effect on IRGM promoter activity, HeLa cells were cotransfected with viral protein 2C or 3C and IRGM-promoter-luc. Luciferase activity was measured 24 h after transfection. The data showed that the relative luciferase activities of the IRGM promoter were markedly promoted in cells over-expressing 2C (P< 0.01), but not in cells over-expressing 3C (Fig 5D). Additionally, we performed a Co-IP assay to detect the physical interaction between IRGM and 2C or 3C (S3C Fig). Regrettably, we failed to confirm the direct interaction between the viral proteins and IRGM. Together, our results suggested that it is possible that 2C could enhance IRGM promoter activation, resulting in increased IRGM expression and the induction of autophagy.

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