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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

Colocalization analyses of aggregated polyhedrin with HSC/HSP70s, ubiquitinated proteins and mitochondria.Infected cells expressing polyhedrin-mCherry and polyhedrin-eGFP were collected at 24 h p.i., treated with paraformaldehyde and Triton X-100, and then immunostained for HSC/HSP70s (A) and ubiquitin (B). The mitochondria (C) were stained with a MitoTracker® deep red probe (Molecular Probes, Life technologies, Eugene, OR, USA). Cell nuclei were stained with Hoechst 33342. Cells were imaged under a Leica TCS SP5 confocal laser-scanning microscope.
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f5: Colocalization analyses of aggregated polyhedrin with HSC/HSP70s, ubiquitinated proteins and mitochondria.Infected cells expressing polyhedrin-mCherry and polyhedrin-eGFP were collected at 24 h p.i., treated with paraformaldehyde and Triton X-100, and then immunostained for HSC/HSP70s (A) and ubiquitin (B). The mitochondria (C) were stained with a MitoTracker® deep red probe (Molecular Probes, Life technologies, Eugene, OR, USA). Cell nuclei were stained with Hoechst 33342. Cells were imaged under a Leica TCS SP5 confocal laser-scanning microscope.

Mentions: Next, whether polyhedrin foci possess other hallmark characteristics of aggresomes was demonstrated. Formation of aggresomes has been reported to recruit cytosolic components such as chaperones, ubiquitinated proteins and mitochondria, to facilitate clearance of aggregated proteins2728. To characterize further that polyhedrin aggregates are aggresomes, association of HSC/HSP70, ubiquitin and mitochondria with polyhedrin was investigated. BmN cells were infected with the virus to highly express fusion protein polyhedrin-mCherry. After treatment by paraformaldehyde and Triton X-100, cells were immunostained for HSC/HSP70 and ubiquitinated proteins. Clearly, a subpopulation of HSC/HSP70s and ubiquitinated proteins colocalized with polyhedrin-mCherry in the cytoplasm to aggregated foci (Fig. 5A,B). Aggregates caused proteotoxicity in cells and they often recruit mitochondria to provide ATP required by chaperones and proteasomes for aggregated protein refolding and/or degradation. To investigate colocalization of mitochondria with polyhedrin aggresome structures, BmN cells were infected with a virus to highly express the fusion protein polyhedrin-eGFP, and then assessed by staining cells with a mitochondrion-selective dye, MitoTracker Red. Results showed these polyhedrin-eGFP foci colocalized with mitochondria (Fig. 5C). These data therefore provide additional evidences that some foci formed by polyhedrin are aggresomes.


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)

Colocalization analyses of aggregated polyhedrin with HSC/HSP70s, ubiquitinated proteins and mitochondria.Infected cells expressing polyhedrin-mCherry and polyhedrin-eGFP were collected at 24 h p.i., treated with paraformaldehyde and Triton X-100, and then immunostained for HSC/HSP70s (A) and ubiquitin (B). The mitochondria (C) were stained with a MitoTracker® deep red probe (Molecular Probes, Life technologies, Eugene, OR, USA). Cell nuclei were stained with Hoechst 33342. Cells were imaged under a Leica TCS SP5 confocal laser-scanning microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Colocalization analyses of aggregated polyhedrin with HSC/HSP70s, ubiquitinated proteins and mitochondria.Infected cells expressing polyhedrin-mCherry and polyhedrin-eGFP were collected at 24 h p.i., treated with paraformaldehyde and Triton X-100, and then immunostained for HSC/HSP70s (A) and ubiquitin (B). The mitochondria (C) were stained with a MitoTracker® deep red probe (Molecular Probes, Life technologies, Eugene, OR, USA). Cell nuclei were stained with Hoechst 33342. Cells were imaged under a Leica TCS SP5 confocal laser-scanning microscope.
Mentions: Next, whether polyhedrin foci possess other hallmark characteristics of aggresomes was demonstrated. Formation of aggresomes has been reported to recruit cytosolic components such as chaperones, ubiquitinated proteins and mitochondria, to facilitate clearance of aggregated proteins2728. To characterize further that polyhedrin aggregates are aggresomes, association of HSC/HSP70, ubiquitin and mitochondria with polyhedrin was investigated. BmN cells were infected with the virus to highly express fusion protein polyhedrin-mCherry. After treatment by paraformaldehyde and Triton X-100, cells were immunostained for HSC/HSP70 and ubiquitinated proteins. Clearly, a subpopulation of HSC/HSP70s and ubiquitinated proteins colocalized with polyhedrin-mCherry in the cytoplasm to aggregated foci (Fig. 5A,B). Aggregates caused proteotoxicity in cells and they often recruit mitochondria to provide ATP required by chaperones and proteasomes for aggregated protein refolding and/or degradation. To investigate colocalization of mitochondria with polyhedrin aggresome structures, BmN cells were infected with a virus to highly express the fusion protein polyhedrin-eGFP, and then assessed by staining cells with a mitochondrion-selective dye, MitoTracker Red. Results showed these polyhedrin-eGFP foci colocalized with mitochondria (Fig. 5C). These data therefore provide additional evidences that some foci formed by polyhedrin are aggresomes.

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