Limits...
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

Inhibition of autophagy with specific siRNA targeting endogenous ATG7 enhances TGEV replication.(a) ST cells were transfected with siRNA targeting porcine endogenous ATG7 or control siRNA for 24 h, and followed by mock treatment or TGEV infection at MOI of 1. At 24 hpi, the silencing efficiency of ATG7 siRNA was analyzed. (b) Detergent lysate from equivalent numbers of ST cells transfected with ATG7-specific siRNA were subjected to immunoblotting with antibodies to ATG7 or β-actin. (c) ST cells were transfected with ATG7-specific siRNA duplexes 2# or control siRNA for 24 h followed by infection with TGEV. At 24 h post infection, TGEV infection was examined by IFA. The percentage of TGEV-infected cells per view from three independent experiments is expressed as mean ± SD. (d) Virus titers in ATG7-siRNA 2#-transfected ST cells were determined by TCID50 assay. Representative data from three independent experiments are shown as the 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. S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814908&req=5

f9: Inhibition of autophagy with specific siRNA targeting endogenous ATG7 enhances TGEV replication.(a) ST cells were transfected with siRNA targeting porcine endogenous ATG7 or control siRNA for 24 h, and followed by mock treatment or TGEV infection at MOI of 1. At 24 hpi, the silencing efficiency of ATG7 siRNA was analyzed. (b) Detergent lysate from equivalent numbers of ST cells transfected with ATG7-specific siRNA were subjected to immunoblotting with antibodies to ATG7 or β-actin. (c) ST cells were transfected with ATG7-specific siRNA duplexes 2# or control siRNA for 24 h followed by infection with TGEV. At 24 h post infection, TGEV infection was examined by IFA. The percentage of TGEV-infected cells per view from three independent experiments is expressed as mean ± SD. (d) Virus titers in ATG7-siRNA 2#-transfected ST cells were determined by TCID50 assay. Representative data from three independent experiments are shown as the 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. S7.

Mentions: In additional experiments with pharmalogical regulators, we used siRNA duplexes that target three highly conserved genes required for mammalian autophagy (LC3, ATG5, and ATG7)313233. These three genes were examined to confirm that the effects observed were due to the autophagy pathway rather than to the non-target effects of the siRNA. To deplete the proteins, we treated ST cells with siRNA duplexes for 24 h and then challenged the cells with TGEV. When cells were separately treated with each of the three siRNA duplexes, the levels of LC3, ATG5, and ATG7 mRNA were decreased in each case relative to levels in cells treated with nontargeting control siRNA (Figs 7a, 8a and 9a). Western blot analysis of detergent lysates collected from cells revealed that levels of endogenous LC3, ATG5, or ATG7 protein were significantly lower in cells treated with the siRNA duplexes than in cells treated with nontargeting control siRNA (Figs 7b, 8b and 9b). Because the data indicated that knockdown efficiency was greatest with LC3-specific siRNA duplexes 3#, ATG5-specific siRNA duplexes 1#, and ATG7-specific siRNA duplexes 2#, these specific siRNA duplexes were used in the following experiments. Following the knockdown of each autophagy-related gene with these gene-specific siRNA duplexes, we observed an increase in the percentage of TGEV-infected cells in comparison to the nontargeting control siRNA (Figs 7c, 8c and 9c). We also found that autophagic protein depletion led to increased viral titers (Figs 7d, 8d and 9d). Together, the data demonstrate that inhibition of autophagy promotes TGEV replication.


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)

Inhibition of autophagy with specific siRNA targeting endogenous ATG7 enhances TGEV replication.(a) ST cells were transfected with siRNA targeting porcine endogenous ATG7 or control siRNA for 24 h, and followed by mock treatment or TGEV infection at MOI of 1. At 24 hpi, the silencing efficiency of ATG7 siRNA was analyzed. (b) Detergent lysate from equivalent numbers of ST cells transfected with ATG7-specific siRNA were subjected to immunoblotting with antibodies to ATG7 or β-actin. (c) ST cells were transfected with ATG7-specific siRNA duplexes 2# or control siRNA for 24 h followed by infection with TGEV. At 24 h post infection, TGEV infection was examined by IFA. The percentage of TGEV-infected cells per view from three independent experiments is expressed as mean ± SD. (d) Virus titers in ATG7-siRNA 2#-transfected ST cells were determined by TCID50 assay. Representative data from three independent experiments are shown as the 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. S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9: Inhibition of autophagy with specific siRNA targeting endogenous ATG7 enhances TGEV replication.(a) ST cells were transfected with siRNA targeting porcine endogenous ATG7 or control siRNA for 24 h, and followed by mock treatment or TGEV infection at MOI of 1. At 24 hpi, the silencing efficiency of ATG7 siRNA was analyzed. (b) Detergent lysate from equivalent numbers of ST cells transfected with ATG7-specific siRNA were subjected to immunoblotting with antibodies to ATG7 or β-actin. (c) ST cells were transfected with ATG7-specific siRNA duplexes 2# or control siRNA for 24 h followed by infection with TGEV. At 24 h post infection, TGEV infection was examined by IFA. The percentage of TGEV-infected cells per view from three independent experiments is expressed as mean ± SD. (d) Virus titers in ATG7-siRNA 2#-transfected ST cells were determined by TCID50 assay. Representative data from three independent experiments are shown as the 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. S7.
Mentions: In additional experiments with pharmalogical regulators, we used siRNA duplexes that target three highly conserved genes required for mammalian autophagy (LC3, ATG5, and ATG7)313233. These three genes were examined to confirm that the effects observed were due to the autophagy pathway rather than to the non-target effects of the siRNA. To deplete the proteins, we treated ST cells with siRNA duplexes for 24 h and then challenged the cells with TGEV. When cells were separately treated with each of the three siRNA duplexes, the levels of LC3, ATG5, and ATG7 mRNA were decreased in each case relative to levels in cells treated with nontargeting control siRNA (Figs 7a, 8a and 9a). Western blot analysis of detergent lysates collected from cells revealed that levels of endogenous LC3, ATG5, or ATG7 protein were significantly lower in cells treated with the siRNA duplexes than in cells treated with nontargeting control siRNA (Figs 7b, 8b and 9b). Because the data indicated that knockdown efficiency was greatest with LC3-specific siRNA duplexes 3#, ATG5-specific siRNA duplexes 1#, and ATG7-specific siRNA duplexes 2#, these specific siRNA duplexes were used in the following experiments. Following the knockdown of each autophagy-related gene with these gene-specific siRNA duplexes, we observed an increase in the percentage of TGEV-infected cells in comparison to the nontargeting control siRNA (Figs 7c, 8c and 9c). We also found that autophagic protein depletion led to increased viral titers (Figs 7d, 8d and 9d). Together, the data demonstrate that inhibition of autophagy promotes TGEV replication.

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