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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 enhances the autophagic flux.(a) ST cells were mock-treated or TGEV-infected at MOI of 1. At the indicated time points post infection, mock- and TGEV-infected cells were lysed and blotted with antibody against p62 or β-actin as indicated. (b) Densitometric data of p62/β-actin in ST cells from three independent experiments are expressed as mean ± SD. *p < 0.05. The p value is calculated using Student’s t-test. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Fig. S3.
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f4: TGEV infection enhances the autophagic flux.(a) ST cells were mock-treated or TGEV-infected at MOI of 1. At the indicated time points post infection, mock- and TGEV-infected cells were lysed and blotted with antibody against p62 or β-actin as indicated. (b) Densitometric data of p62/β-actin in ST cells from three independent experiments are expressed as mean ± SD. *p < 0.05. The p value is calculated using Student’s t-test. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Fig. S3.

Mentions: To determine whether autophagosome accumulation is due to autophagy induction or to a block in the maturation of autophagosome into autolysosomes2425, we performed autophagic flux assays, which can distinguish between these two possibilities26. Because p62/SQSTM1 serves as a link between LC3 and ubiquitinated substrates, and because inhibition of autophagy leads to an increase in the levels of p62, p62 degradation is considered an indicator of autophagic flux2227. Therefore, we used a western blot assay to measure the changes in p62 protein levels upon virus infection. As shown in Fig. 4a,b, TGEV infection increased degradation of p62 in ST cells over time. There was no significant alteration of p62 between 0 h and 36 h in the mock-treated control. These data indicate that the accumulation of autophagosomes induced by TGEV infection is due to autophagic activation rather than to a block in autophagosome maturation.


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 enhances the autophagic flux.(a) ST cells were mock-treated or TGEV-infected at MOI of 1. At the indicated time points post infection, mock- and TGEV-infected cells were lysed and blotted with antibody against p62 or β-actin as indicated. (b) Densitometric data of p62/β-actin in ST cells from three independent experiments are expressed as mean ± SD. *p < 0.05. The p value is calculated using Student’s t-test. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Fig. S3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: TGEV infection enhances the autophagic flux.(a) ST cells were mock-treated or TGEV-infected at MOI of 1. At the indicated time points post infection, mock- and TGEV-infected cells were lysed and blotted with antibody against p62 or β-actin as indicated. (b) Densitometric data of p62/β-actin in ST cells from three independent experiments are expressed as mean ± SD. *p < 0.05. The p value is calculated using Student’s t-test. Gels were run under the same experimental conditions. For better clarity and conciseness of the presentation, cropped blots are shown. The raw uncropped images can be found in the Supplementary Fig. S3.
Mentions: To determine whether autophagosome accumulation is due to autophagy induction or to a block in the maturation of autophagosome into autolysosomes2425, we performed autophagic flux assays, which can distinguish between these two possibilities26. Because p62/SQSTM1 serves as a link between LC3 and ubiquitinated substrates, and because inhibition of autophagy leads to an increase in the levels of p62, p62 degradation is considered an indicator of autophagic flux2227. Therefore, we used a western blot assay to measure the changes in p62 protein levels upon virus infection. As shown in Fig. 4a,b, TGEV infection increased degradation of p62 in ST cells over time. There was no significant alteration of p62 between 0 h and 36 h in the mock-treated control. These data indicate that the accumulation of autophagosomes induced by TGEV infection is due to autophagic activation rather than to a block in autophagosome maturation.

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