Limits...
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

Higher apoptosis rates in Hep-dG-infected NA cells. (A) Effects of rabies viruses rHep-Flury and Hep-dG on cell viability. NA cells were infected with rHep-Flury and Hep-dG at various MOI of 0.01, 0.1, and 1. Cell viability was assessed with the cell counting kit-8. Absorbance at 450 nm was measured using a microplate reader. Data were mean ± SEM. n = 6. Differences (independent-samples t-Test) being **p < 0.01, and ***p < 0.001. (B) Cell apoptosis induced by rabies viruses Hep-dG and rHep-Flury. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. Cell apoptosis was quantified by using Annexin V-FITC apoptosis kit. Samples were analyzed by flow cytometry. The lower right part (Annexin V+/PI-) was considered as early stage of apoptotic cells and top right part (Annexin V-/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-/PI-) was considered as viable cells and the upper left part (Annexin V-/PI+) was considered as necrotic cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493837&req=5

Figure 2: Higher apoptosis rates in Hep-dG-infected NA cells. (A) Effects of rabies viruses rHep-Flury and Hep-dG on cell viability. NA cells were infected with rHep-Flury and Hep-dG at various MOI of 0.01, 0.1, and 1. Cell viability was assessed with the cell counting kit-8. Absorbance at 450 nm was measured using a microplate reader. Data were mean ± SEM. n = 6. Differences (independent-samples t-Test) being **p < 0.01, and ***p < 0.001. (B) Cell apoptosis induced by rabies viruses Hep-dG and rHep-Flury. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. Cell apoptosis was quantified by using Annexin V-FITC apoptosis kit. Samples were analyzed by flow cytometry. The lower right part (Annexin V+/PI-) was considered as early stage of apoptotic cells and top right part (Annexin V-/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-/PI-) was considered as viable cells and the upper left part (Annexin V-/PI+) was considered as necrotic cells.

Mentions: Cell viability was assessed using the CCK-8 assay. NA cells infected with either rabies virus showed a dose-dependent decrease in cell viability (Figure 2A). However, the viability of Hep-dG-infected cells was extremely and significantly reduced compared with the rHep-Flury group at various MOI of 0.01, 0.1, and 1 (P < 0.001, independent sample t-test using SPSS). Interestingly, at a very low MOI of 0.01, Hep-dG-infected cells showed a significant decrease in the viability, while rHep-Flury did not cause toxicity in these conditions. Since infection of Hep-dG induces significant cell death at MOI of 0. 1 or 1, MOI = 0.01 was chosen in the iTRAQ experiment.


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)

Higher apoptosis rates in Hep-dG-infected NA cells. (A) Effects of rabies viruses rHep-Flury and Hep-dG on cell viability. NA cells were infected with rHep-Flury and Hep-dG at various MOI of 0.01, 0.1, and 1. Cell viability was assessed with the cell counting kit-8. Absorbance at 450 nm was measured using a microplate reader. Data were mean ± SEM. n = 6. Differences (independent-samples t-Test) being **p < 0.01, and ***p < 0.001. (B) Cell apoptosis induced by rabies viruses Hep-dG and rHep-Flury. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. Cell apoptosis was quantified by using Annexin V-FITC apoptosis kit. Samples were analyzed by flow cytometry. The lower right part (Annexin V+/PI-) was considered as early stage of apoptotic cells and top right part (Annexin V-/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-/PI-) was considered as viable cells and the upper left part (Annexin V-/PI+) was considered as necrotic cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Higher apoptosis rates in Hep-dG-infected NA cells. (A) Effects of rabies viruses rHep-Flury and Hep-dG on cell viability. NA cells were infected with rHep-Flury and Hep-dG at various MOI of 0.01, 0.1, and 1. Cell viability was assessed with the cell counting kit-8. Absorbance at 450 nm was measured using a microplate reader. Data were mean ± SEM. n = 6. Differences (independent-samples t-Test) being **p < 0.01, and ***p < 0.001. (B) Cell apoptosis induced by rabies viruses Hep-dG and rHep-Flury. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. Cell apoptosis was quantified by using Annexin V-FITC apoptosis kit. Samples were analyzed by flow cytometry. The lower right part (Annexin V+/PI-) was considered as early stage of apoptotic cells and top right part (Annexin V-/PI+) was considered as late stage of apoptotic cells. The lower left part (Annexin V-/PI-) was considered as viable cells and the upper left part (Annexin V-/PI+) was considered as necrotic cells.
Mentions: Cell viability was assessed using the CCK-8 assay. NA cells infected with either rabies virus showed a dose-dependent decrease in cell viability (Figure 2A). However, the viability of Hep-dG-infected cells was extremely and significantly reduced compared with the rHep-Flury group at various MOI of 0.01, 0.1, and 1 (P < 0.001, independent sample t-test using SPSS). Interestingly, at a very low MOI of 0.01, Hep-dG-infected cells showed a significant decrease in the viability, while rHep-Flury did not cause toxicity in these conditions. Since infection of Hep-dG induces significant cell death at MOI of 0. 1 or 1, MOI = 0.01 was chosen in the iTRAQ experiment.

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