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Characterization of aggregate/aggresome structures formed by polyhedrin of Bombyx mori nucleopolyhedrovirus.

Guo ZJ, Tao LX, Dong XY, Yu MH, Tian T, Tang XD - Sci Rep (2015)

Bottom Line: At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy.Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production.These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, Jiangsu, P.R. China.

ABSTRACT
Virus infections often lead to formation of aggregates and aggresomes in host cells. In this study, production of aggregates and aggresomes by the highly expressed protein polyhedrin of Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h postinfection (p.i.) was detected with a fluorescent molecular dye, and verified by colocalization of polyhedrin with aggresomal markers, GFP-250 and γ-tubulin. Polyhedrin aggregates showed hallmark characteristics of aggresomes: formation was microtubule-dependent; they colocalized with heat shock cognates/proteins of the 70-kDa family (HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria. Aggregated polyhedrin protein gradually gained its active conformation accompanying progress of BmNPV infection. At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production. These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

No MeSH data available.


Related in: MedlinePlus

Polyhedrin, the protein expressed in a large amount, formed aggregates/aggresomes.(A) Colocalization between polyhedrin and aggregates/aggresomes in the cytoplasm. Cells were infected with BmNPV T3 isolate at an MOI of 10 TCID50/cell, and at selected time points fixed and permeabilized, then incubated in the blocking buffer, followed by incubation with mouse monoclonal anti-polyhedrin antibody and FITC-conjugated goat anti-mouse IgG. Next, cells were stained with ProteoStat® dye and Hoechst 33342 prepared according to kit instructions for 30 min at room temperature, washed with 1 × PBS and then imaged by a fluorescence microscopy using a Texas Red filter set for the ProteoStat® dye, and an FITC filter set for FITC-conjugated antibody respectively. (B) Percentage of cells displaying colocalization between aggregates/aggresomes and polyhedrin in the cytoplasm. Each percentage value is obtained from three independent infections, and for each infection, 90–120 cells were counted. Error bars indicates ±SEM. (C) Fusion protein polyhedrin-mCherry aggregated (foci) in the cytoplasm at 24 h p.i. Polyhedrin-mCherry was expressed using the BmNPV Bac-to-Bac system then visualized under a confocal laser scanning microscope at 24 and 48 h p.i. (D) Fluorescent proteins eGFP and mCherry alone do not localize to cytoplasmic foci. Plasmids pPie1-eGFP, pPph-eGFP and pPph-mCherry (Supplementary Table S2) were transposed. Positive bacmids were transfected into BmN cells to obtain BV which was used for infection. Fluorescent cells were visualized at 24 or 48 h p.i.
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f3: Polyhedrin, the protein expressed in a large amount, formed aggregates/aggresomes.(A) Colocalization between polyhedrin and aggregates/aggresomes in the cytoplasm. Cells were infected with BmNPV T3 isolate at an MOI of 10 TCID50/cell, and at selected time points fixed and permeabilized, then incubated in the blocking buffer, followed by incubation with mouse monoclonal anti-polyhedrin antibody and FITC-conjugated goat anti-mouse IgG. Next, cells were stained with ProteoStat® dye and Hoechst 33342 prepared according to kit instructions for 30 min at room temperature, washed with 1 × PBS and then imaged by a fluorescence microscopy using a Texas Red filter set for the ProteoStat® dye, and an FITC filter set for FITC-conjugated antibody respectively. (B) Percentage of cells displaying colocalization between aggregates/aggresomes and polyhedrin in the cytoplasm. Each percentage value is obtained from three independent infections, and for each infection, 90–120 cells were counted. Error bars indicates ±SEM. (C) Fusion protein polyhedrin-mCherry aggregated (foci) in the cytoplasm at 24 h p.i. Polyhedrin-mCherry was expressed using the BmNPV Bac-to-Bac system then visualized under a confocal laser scanning microscope at 24 and 48 h p.i. (D) Fluorescent proteins eGFP and mCherry alone do not localize to cytoplasmic foci. Plasmids pPie1-eGFP, pPph-eGFP and pPph-mCherry (Supplementary Table S2) were transposed. Positive bacmids were transfected into BmN cells to obtain BV which was used for infection. Fluorescent cells were visualized at 24 or 48 h p.i.

Mentions: As described above, infection of BmNPV could induce formation of aggregates and aggresomes, raising the question of where these toxic structures come from. During BmNPV progeny production, a large number of viral proteins, e.g. polyhedrin, are synthesized in a relatively short time and a large amount, whereby protein folding can become a limiting step for their active conformation and trafficking. It was inferred that some proteins encoded by BmNPV genome formed aggregates and aggresomes. Firstly, to address it, the fibrillation propensity profiles within polyhedrin amino acid sequence were assessed by a 3D profile method. Energy per amino acid below a threshold value of –23 kcal/mol was indicative of a high fibrillation propensity. Segments obtained from 3D profile method were showed in the image where the aggregation-prone regions were found to be Gly25-Cys26, Asp81-Lys84, Glu186-Tyr187 and Glu219-Val224 (Fig. 2). Then co-staining of aggregates/aggresomes for polyhedrin was performed. BmN cells were infected, fixed and permeabilized at 24 h p.i., and then stained with a mouse monoclonal anti-polyhedrin antibody and the ProteoStat® dye. As shown in Fig. 3A (upper panel), colocalization between aggregates/aggresomes and polyhedrin was evident at 24 h p.i.


Characterization of aggregate/aggresome structures formed by polyhedrin of Bombyx mori nucleopolyhedrovirus.

Guo ZJ, Tao LX, Dong XY, Yu MH, Tian T, Tang XD - Sci Rep (2015)

Polyhedrin, the protein expressed in a large amount, formed aggregates/aggresomes.(A) Colocalization between polyhedrin and aggregates/aggresomes in the cytoplasm. Cells were infected with BmNPV T3 isolate at an MOI of 10 TCID50/cell, and at selected time points fixed and permeabilized, then incubated in the blocking buffer, followed by incubation with mouse monoclonal anti-polyhedrin antibody and FITC-conjugated goat anti-mouse IgG. Next, cells were stained with ProteoStat® dye and Hoechst 33342 prepared according to kit instructions for 30 min at room temperature, washed with 1 × PBS and then imaged by a fluorescence microscopy using a Texas Red filter set for the ProteoStat® dye, and an FITC filter set for FITC-conjugated antibody respectively. (B) Percentage of cells displaying colocalization between aggregates/aggresomes and polyhedrin in the cytoplasm. Each percentage value is obtained from three independent infections, and for each infection, 90–120 cells were counted. Error bars indicates ±SEM. (C) Fusion protein polyhedrin-mCherry aggregated (foci) in the cytoplasm at 24 h p.i. Polyhedrin-mCherry was expressed using the BmNPV Bac-to-Bac system then visualized under a confocal laser scanning microscope at 24 and 48 h p.i. (D) Fluorescent proteins eGFP and mCherry alone do not localize to cytoplasmic foci. Plasmids pPie1-eGFP, pPph-eGFP and pPph-mCherry (Supplementary Table S2) were transposed. Positive bacmids were transfected into BmN cells to obtain BV which was used for infection. Fluorescent cells were visualized at 24 or 48 h p.i.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4594129&req=5

f3: Polyhedrin, the protein expressed in a large amount, formed aggregates/aggresomes.(A) Colocalization between polyhedrin and aggregates/aggresomes in the cytoplasm. Cells were infected with BmNPV T3 isolate at an MOI of 10 TCID50/cell, and at selected time points fixed and permeabilized, then incubated in the blocking buffer, followed by incubation with mouse monoclonal anti-polyhedrin antibody and FITC-conjugated goat anti-mouse IgG. Next, cells were stained with ProteoStat® dye and Hoechst 33342 prepared according to kit instructions for 30 min at room temperature, washed with 1 × PBS and then imaged by a fluorescence microscopy using a Texas Red filter set for the ProteoStat® dye, and an FITC filter set for FITC-conjugated antibody respectively. (B) Percentage of cells displaying colocalization between aggregates/aggresomes and polyhedrin in the cytoplasm. Each percentage value is obtained from three independent infections, and for each infection, 90–120 cells were counted. Error bars indicates ±SEM. (C) Fusion protein polyhedrin-mCherry aggregated (foci) in the cytoplasm at 24 h p.i. Polyhedrin-mCherry was expressed using the BmNPV Bac-to-Bac system then visualized under a confocal laser scanning microscope at 24 and 48 h p.i. (D) Fluorescent proteins eGFP and mCherry alone do not localize to cytoplasmic foci. Plasmids pPie1-eGFP, pPph-eGFP and pPph-mCherry (Supplementary Table S2) were transposed. Positive bacmids were transfected into BmN cells to obtain BV which was used for infection. Fluorescent cells were visualized at 24 or 48 h p.i.
Mentions: As described above, infection of BmNPV could induce formation of aggregates and aggresomes, raising the question of where these toxic structures come from. During BmNPV progeny production, a large number of viral proteins, e.g. polyhedrin, are synthesized in a relatively short time and a large amount, whereby protein folding can become a limiting step for their active conformation and trafficking. It was inferred that some proteins encoded by BmNPV genome formed aggregates and aggresomes. Firstly, to address it, the fibrillation propensity profiles within polyhedrin amino acid sequence were assessed by a 3D profile method. Energy per amino acid below a threshold value of –23 kcal/mol was indicative of a high fibrillation propensity. Segments obtained from 3D profile method were showed in the image where the aggregation-prone regions were found to be Gly25-Cys26, Asp81-Lys84, Glu186-Tyr187 and Glu219-Val224 (Fig. 2). Then co-staining of aggregates/aggresomes for polyhedrin was performed. BmN cells were infected, fixed and permeabilized at 24 h p.i., and then stained with a mouse monoclonal anti-polyhedrin antibody and the ProteoStat® dye. As shown in Fig. 3A (upper panel), colocalization between aggregates/aggresomes and polyhedrin was evident at 24 h p.i.

Bottom Line: At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy.Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production.These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, Jiangsu, P.R. China.

ABSTRACT
Virus infections often lead to formation of aggregates and aggresomes in host cells. In this study, production of aggregates and aggresomes by the highly expressed protein polyhedrin of Bombyx mori nucleopolyhedrovirus (BmNPV) at 24 h postinfection (p.i.) was detected with a fluorescent molecular dye, and verified by colocalization of polyhedrin with aggresomal markers, GFP-250 and γ-tubulin. Polyhedrin aggregates showed hallmark characteristics of aggresomes: formation was microtubule-dependent; they colocalized with heat shock cognates/proteins of the 70-kDa family (HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria. Aggregated polyhedrin protein gradually gained its active conformation accompanying progress of BmNPV infection. At 48 h p.i. recovered polyhedrin bound directly to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and was colocalized with BmLC3 to the isolation membrane of autophagosome, implying the involvement of polyhedrin in cellular autophagy. Inhibition of autophagy by 3-methyladenine (3-MA) dramatically resulted in decrease of polyhedrin expression and polyhedra particle production. These observations suggested that highly expressed polyhedrin forms aggregate to get involved in cellular autophagy then play an important role in polyhedra production.

No MeSH data available.


Related in: MedlinePlus