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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

Lower virus titers and higher G protein expression levels in Hep-dG-infected NA cells. (A) The replication of rabies viruses rHep-Flury and Hep-dG in NA cells. NA cells were infected with rHep-Flury (■) and Hep-dG (•) at a MOI of 0.01. Supernatants were harvested at 24, 48, 72, and 96 h post-infection, and virus titers were determined by fluorescent-focus assay. Data were mean ± SEM. n = 3. (B) Rabies virus structural protein in NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). (C) Virus infection rate in NA Cells. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. After 24, 48, 72, and 96 h of viral inoculation, FITC anti-rabies monoclonal globulin was used for staining. Images were taken at an original magnification of 200×.
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Figure 1: Lower virus titers and higher G protein expression levels in Hep-dG-infected NA cells. (A) The replication of rabies viruses rHep-Flury and Hep-dG in NA cells. NA cells were infected with rHep-Flury (■) and Hep-dG (•) at a MOI of 0.01. Supernatants were harvested at 24, 48, 72, and 96 h post-infection, and virus titers were determined by fluorescent-focus assay. Data were mean ± SEM. n = 3. (B) Rabies virus structural protein in NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). (C) Virus infection rate in NA Cells. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. After 24, 48, 72, and 96 h of viral inoculation, FITC anti-rabies monoclonal globulin was used for staining. Images were taken at an original magnification of 200×.

Mentions: The replication of RABV rHep-Flury and Hep-dG in NA cells was examined by single step growth curves. As shown in Figure 1A, the virus titers of the rHep-Flury were higher than those obtained for Hep-dG at each time point. Indeed, maximum virus titers of rHep-Flury and Hep-dG were 1 × 107.25 and 1 × 106.10 FFU/ml in NA cells, respectively. At 24, 48, 72, and 96 h post-infection, the titers of the rHep-Flury virus in NA cells were significantly higher than those of Hep-dG virus (p < 0.05, independent sample t-test using the Statistical Package for Social Sciences (SPSS), version 17.0).


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)

Lower virus titers and higher G protein expression levels in Hep-dG-infected NA cells. (A) The replication of rabies viruses rHep-Flury and Hep-dG in NA cells. NA cells were infected with rHep-Flury (■) and Hep-dG (•) at a MOI of 0.01. Supernatants were harvested at 24, 48, 72, and 96 h post-infection, and virus titers were determined by fluorescent-focus assay. Data were mean ± SEM. n = 3. (B) Rabies virus structural protein in NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). (C) Virus infection rate in NA Cells. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. After 24, 48, 72, and 96 h of viral inoculation, FITC anti-rabies monoclonal globulin was used for staining. Images were taken at an original magnification of 200×.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493837&req=5

Figure 1: Lower virus titers and higher G protein expression levels in Hep-dG-infected NA cells. (A) The replication of rabies viruses rHep-Flury and Hep-dG in NA cells. NA cells were infected with rHep-Flury (■) and Hep-dG (•) at a MOI of 0.01. Supernatants were harvested at 24, 48, 72, and 96 h post-infection, and virus titers were determined by fluorescent-focus assay. Data were mean ± SEM. n = 3. (B) Rabies virus structural protein in NA cells were quantified by western blotting. Densitometry of the western blotting was analyzed with the Image J software (NIH). (C) Virus infection rate in NA Cells. NA cells were infected with rHep-Flury and Hep-dG at a MOI of 0.01. After 24, 48, 72, and 96 h of viral inoculation, FITC anti-rabies monoclonal globulin was used for staining. Images were taken at an original magnification of 200×.
Mentions: The replication of RABV rHep-Flury and Hep-dG in NA cells was examined by single step growth curves. As shown in Figure 1A, the virus titers of the rHep-Flury were higher than those obtained for Hep-dG at each time point. Indeed, maximum virus titers of rHep-Flury and Hep-dG were 1 × 107.25 and 1 × 106.10 FFU/ml in NA cells, respectively. At 24, 48, 72, and 96 h post-infection, the titers of the rHep-Flury virus in NA cells were significantly higher than those of Hep-dG virus (p < 0.05, independent sample t-test using the Statistical Package for Social Sciences (SPSS), version 17.0).

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