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Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

Guo L, Yu H, Gu W, Luo X, Li R, Zhang J, Xu Y, Yang L, Shen N, Feng L, Wang Y - Sci Rep (2016)

Bottom Line: In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy.The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy.Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

ABSTRACT
Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

No MeSH data available.


Related in: MedlinePlus

TGEV Infection leads to autophagic vesicle formation.ST cells (a) and PK15 cells (b) were mock-treated or infected with TGEV H165 at MOI of 1 for 24 h. Then the mock- and TGEV-treated cells were fixed and processed for electron microscopy analysis. The vesicles with the characteristics of autophagosome are indicated by black arrows in the relevant parts. Scale bars, 500 nm. (c) Quantification of the number of autophagosome-like vesicles per cell profile in mock- and TGEV-treated cells. Error bars, mean ± SD for 20 cells per experimental condition of three independent experiments. *p < 0.05. The p value is calculated using Student’s t-test.
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f1: TGEV Infection leads to autophagic vesicle formation.ST cells (a) and PK15 cells (b) were mock-treated or infected with TGEV H165 at MOI of 1 for 24 h. Then the mock- and TGEV-treated cells were fixed and processed for electron microscopy analysis. The vesicles with the characteristics of autophagosome are indicated by black arrows in the relevant parts. Scale bars, 500 nm. (c) Quantification of the number of autophagosome-like vesicles per cell profile in mock- and TGEV-treated cells. Error bars, mean ± SD for 20 cells per experimental condition of three independent experiments. *p < 0.05. The p value is calculated using Student’s t-test.

Mentions: Upon infection, the betacoronavirus MHV uses components of the cellular autophagic pathway to form DMVs in order to complete viral replication18. To determine whether TGEV infection regulates the cellular autophagy process by inducing the formation of DMVs, we used transmission electron microscopy (TEM) to examine the formation of autophagosome-like vesicles in TGEV-infected ST and PK15 cells. We observed that the number of double- and single-membrane vesicles increased near the perinuclear region of TGEV-infected ST cells, and that such structures were rare in the mock-treated samples (Fig. 1a). These autophagosome-like vesicles were morphologically identical to the MHV-infected Hela cells6. Similar results were obtained using PK15 cells (Fig. 1b). The number of autophagosome-like vesicles was greater in the cytoplasm of TGEV-infected cells than in the cytoplasm of mock-treated cells (Fig. 1c).


Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

Guo L, Yu H, Gu W, Luo X, Li R, Zhang J, Xu Y, Yang L, Shen N, Feng L, Wang Y - Sci Rep (2016)

TGEV Infection leads to autophagic vesicle formation.ST cells (a) and PK15 cells (b) were mock-treated or infected with TGEV H165 at MOI of 1 for 24 h. Then the mock- and TGEV-treated cells were fixed and processed for electron microscopy analysis. The vesicles with the characteristics of autophagosome are indicated by black arrows in the relevant parts. Scale bars, 500 nm. (c) Quantification of the number of autophagosome-like vesicles per cell profile in mock- and TGEV-treated cells. Error bars, mean ± SD for 20 cells per experimental condition of three independent experiments. *p < 0.05. The p value is calculated using Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814908&req=5

f1: TGEV Infection leads to autophagic vesicle formation.ST cells (a) and PK15 cells (b) were mock-treated or infected with TGEV H165 at MOI of 1 for 24 h. Then the mock- and TGEV-treated cells were fixed and processed for electron microscopy analysis. The vesicles with the characteristics of autophagosome are indicated by black arrows in the relevant parts. Scale bars, 500 nm. (c) Quantification of the number of autophagosome-like vesicles per cell profile in mock- and TGEV-treated cells. Error bars, mean ± SD for 20 cells per experimental condition of three independent experiments. *p < 0.05. The p value is calculated using Student’s t-test.
Mentions: Upon infection, the betacoronavirus MHV uses components of the cellular autophagic pathway to form DMVs in order to complete viral replication18. To determine whether TGEV infection regulates the cellular autophagy process by inducing the formation of DMVs, we used transmission electron microscopy (TEM) to examine the formation of autophagosome-like vesicles in TGEV-infected ST and PK15 cells. We observed that the number of double- and single-membrane vesicles increased near the perinuclear region of TGEV-infected ST cells, and that such structures were rare in the mock-treated samples (Fig. 1a). These autophagosome-like vesicles were morphologically identical to the MHV-infected Hela cells6. Similar results were obtained using PK15 cells (Fig. 1b). The number of autophagosome-like vesicles was greater in the cytoplasm of TGEV-infected cells than in the cytoplasm of mock-treated cells (Fig. 1c).

Bottom Line: In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy.The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy.Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China.

ABSTRACT
Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

No MeSH data available.


Related in: MedlinePlus