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iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG.

Yang Y, Liu W, Yan G, Luo Y, Zhao J, Yang X, Mei M, Wu X, Guo X - Front Microbiol (2015)

Bottom Line: Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling.Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner.These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University Guangzhou, China.

ABSTRACT
The rabies virus (RABV) glycoprotein (G) is the principal contributor to the pathogenicity and protective immunity of RABV. In a previous work, we reported that recombinant rabies virus Hep-dG, which was generated by reverse genetics to carry two copies of the G-gene, showed lower virulence than the parental virus rHep-Flury in suckling mice with a better immune protection effect. To better understand the mechanisms underlying rabies virus attenuation and the role of glycoprotein G, isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify and quantify distinct proteins. 10 and 111 differentially expressed proteins were obtained in rHep-Flury and Hep-dG infection groups, respectively. Selected data were validated by western blot and qRT-PCR. Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling. Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner. These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

No MeSH data available.


Related in: MedlinePlus

Validation of selected data by western blot and qRT-PCR. (A) Representative proteins in infected and mock-infected NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). Values are expressed relative to GADPH. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being *p < 0.05, **p < 0.01, and ***p < 0.001. (B) p-STAT1 (T701), p-STAT3 (T705) and autophagy-related markers LC3 in NA cells were quantified by western blotting. (C) IFN gene expression in rabies viruses rHep-Flury and Hep-dG-infected NA cells. GAPDH was used as the internal loading control. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being ***p < 0.001.
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Figure 7: Validation of selected data by western blot and qRT-PCR. (A) Representative proteins in infected and mock-infected NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). Values are expressed relative to GADPH. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being *p < 0.05, **p < 0.01, and ***p < 0.001. (B) p-STAT1 (T701), p-STAT3 (T705) and autophagy-related markers LC3 in NA cells were quantified by western blotting. (C) IFN gene expression in rabies viruses rHep-Flury and Hep-dG-infected NA cells. GAPDH was used as the internal loading control. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being ***p < 0.001.

Mentions: To confirm the differentially expressed proteins identified by the iTRAQ method, several up-regulated and unaltered proteins between infected and mock-infected cells were quantified by western blotting. As shown in Figure 7A, the levels of seven representative proteins, including GAPDH, ISG15, RIG-I/Ddx58, STAT1, and STAT3, were consistent with iTRAQ results.


iTRAQ protein profile analysis of neuroblastoma (NA) cells infected with the rabies viruses rHep-Flury and Hep-dG.

Yang Y, Liu W, Yan G, Luo Y, Zhao J, Yang X, Mei M, Wu X, Guo X - Front Microbiol (2015)

Validation of selected data by western blot and qRT-PCR. (A) Representative proteins in infected and mock-infected NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). Values are expressed relative to GADPH. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being *p < 0.05, **p < 0.01, and ***p < 0.001. (B) p-STAT1 (T701), p-STAT3 (T705) and autophagy-related markers LC3 in NA cells were quantified by western blotting. (C) IFN gene expression in rabies viruses rHep-Flury and Hep-dG-infected NA cells. GAPDH was used as the internal loading control. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493837&req=5

Figure 7: Validation of selected data by western blot and qRT-PCR. (A) Representative proteins in infected and mock-infected NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). Values are expressed relative to GADPH. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being *p < 0.05, **p < 0.01, and ***p < 0.001. (B) p-STAT1 (T701), p-STAT3 (T705) and autophagy-related markers LC3 in NA cells were quantified by western blotting. (C) IFN gene expression in rabies viruses rHep-Flury and Hep-dG-infected NA cells. GAPDH was used as the internal loading control. Data are mean ± SEM. n = 3. Differences (independent-samples t-test) being ***p < 0.001.
Mentions: To confirm the differentially expressed proteins identified by the iTRAQ method, several up-regulated and unaltered proteins between infected and mock-infected cells were quantified by western blotting. As shown in Figure 7A, the levels of seven representative proteins, including GAPDH, ISG15, RIG-I/Ddx58, STAT1, and STAT3, were consistent with iTRAQ results.

Bottom Line: Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling.Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner.These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine, South China Agricultural University Guangzhou, China.

ABSTRACT
The rabies virus (RABV) glycoprotein (G) is the principal contributor to the pathogenicity and protective immunity of RABV. In a previous work, we reported that recombinant rabies virus Hep-dG, which was generated by reverse genetics to carry two copies of the G-gene, showed lower virulence than the parental virus rHep-Flury in suckling mice with a better immune protection effect. To better understand the mechanisms underlying rabies virus attenuation and the role of glycoprotein G, isobaric tags for relative and absolute quantitation (iTRAQ) was performed to identify and quantify distinct proteins. 10 and 111 differentially expressed proteins were obtained in rHep-Flury and Hep-dG infection groups, respectively. Selected data were validated by western blot and qRT-PCR. Bioinformatics analysis of the distinct protein suggested that glycoprotein over-expression in the attenuated RABV strain can induce activation of the interferon signaling. Furthermore, it may promote the antiviral response, MHC-I mediated antigen-specific T cell immune response, apoptosis and autophagy in an IFN-dependent manner. These findings might not only improve the understanding of the dynamics of RABV and host interaction, but also help understand the mechanisms underlying innate and adaptive immunity during RABV infection.

No MeSH data available.


Related in: MedlinePlus