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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-induced autophagy is dependent of viral replication.(a) Immunofluorescence assay verified that UV-inactivated TGEV was replication defective. TGEV N protein was stained with mAb 3D7 and followed by FITC-conjugated goat anti-mouse IgG (green, left column). Nuclei counterstained with DAPI are visualized in blue (middle column). The right column illustrates a merged image of staining with mAb 3D7 and DAPI. (b) ST cells and PK15 cells were inoculated with replication competent TGEV or UV- inactivated TGEV at MOI of 10 for 24 h. Cells were lysed and analyzed by western blot with antibodies as indicated. (c) Densitometric data of LC3-II/LC3-I 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. S2.
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f3: TGEV infection-induced autophagy is dependent of viral replication.(a) Immunofluorescence assay verified that UV-inactivated TGEV was replication defective. TGEV N protein was stained with mAb 3D7 and followed by FITC-conjugated goat anti-mouse IgG (green, left column). Nuclei counterstained with DAPI are visualized in blue (middle column). The right column illustrates a merged image of staining with mAb 3D7 and DAPI. (b) ST cells and PK15 cells were inoculated with replication competent TGEV or UV- inactivated TGEV at MOI of 10 for 24 h. Cells were lysed and analyzed by western blot with antibodies as indicated. (c) Densitometric data of LC3-II/LC3-I 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. S2.

Mentions: The activation of autophagic activity by TGEV infection could be caused either by incoming virons or by viral replication products. To determine whether TGEV replication is required for the induction of autophagy, we challenged cells with either replication-competent or UV-inactivated virus and measured the effect on autophagy by monitoring the conversion of LC3-I to LC3-II. Before conducting the formal experiments, we used immunofluorescence assay (IFA) to verify that the UV-inactivated virus was replication defective (Fig. 3a). By using western blot, we found that the levels of both LC3-I and LC3-II were similar in ST cells inoculated with UV-inactivated TGEV at MOI of 10 and in mock-treated ST cells at 24 h post inoculation; in addition, no N protein synthesis was detected in ST cells inoculated with UV-inactivated TGEV. In contrast, ST cells infected with replication-competent TGEV apparently underwent the conversion of LC3-I to LC3-II (Fig. 3b,c). Similar results were obtained with PK15 cells (Fig. 3b,c), suggesting that TGEV replication is required for the formation of autophagosomes.


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-induced autophagy is dependent of viral replication.(a) Immunofluorescence assay verified that UV-inactivated TGEV was replication defective. TGEV N protein was stained with mAb 3D7 and followed by FITC-conjugated goat anti-mouse IgG (green, left column). Nuclei counterstained with DAPI are visualized in blue (middle column). The right column illustrates a merged image of staining with mAb 3D7 and DAPI. (b) ST cells and PK15 cells were inoculated with replication competent TGEV or UV- inactivated TGEV at MOI of 10 for 24 h. Cells were lysed and analyzed by western blot with antibodies as indicated. (c) Densitometric data of LC3-II/LC3-I 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. S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: TGEV infection-induced autophagy is dependent of viral replication.(a) Immunofluorescence assay verified that UV-inactivated TGEV was replication defective. TGEV N protein was stained with mAb 3D7 and followed by FITC-conjugated goat anti-mouse IgG (green, left column). Nuclei counterstained with DAPI are visualized in blue (middle column). The right column illustrates a merged image of staining with mAb 3D7 and DAPI. (b) ST cells and PK15 cells were inoculated with replication competent TGEV or UV- inactivated TGEV at MOI of 10 for 24 h. Cells were lysed and analyzed by western blot with antibodies as indicated. (c) Densitometric data of LC3-II/LC3-I 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. S2.
Mentions: The activation of autophagic activity by TGEV infection could be caused either by incoming virons or by viral replication products. To determine whether TGEV replication is required for the induction of autophagy, we challenged cells with either replication-competent or UV-inactivated virus and measured the effect on autophagy by monitoring the conversion of LC3-I to LC3-II. Before conducting the formal experiments, we used immunofluorescence assay (IFA) to verify that the UV-inactivated virus was replication defective (Fig. 3a). By using western blot, we found that the levels of both LC3-I and LC3-II were similar in ST cells inoculated with UV-inactivated TGEV at MOI of 10 and in mock-treated ST cells at 24 h post inoculation; in addition, no N protein synthesis was detected in ST cells inoculated with UV-inactivated TGEV. In contrast, ST cells infected with replication-competent TGEV apparently underwent the conversion of LC3-I to LC3-II (Fig. 3b,c). Similar results were obtained with PK15 cells (Fig. 3b,c), suggesting that TGEV replication is required for the formation of autophagosomes.

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