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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

Cellular autophagy played a role in polyhedra particle production.Land M, protein marker. (A) Cellular autophagy was inhibited by 3-MA. (B) Light microscopy observations at 72 h p.i. of 3-MA-treated and untreated BmN cells infected with BmNPV T3 isolate. (C) Percentage of cells displaying OBs in the nucleus. Each value is calculated at 72 h p.i. from three independent infections and for each infection, 298–360 cells were scored. Error bars indicates ±SEM. (D) Western blot analysis of polyhedrin with a mouse monoclonal anti-polyhedrin antibody.
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f8: Cellular autophagy played a role in polyhedra particle production.Land M, protein marker. (A) Cellular autophagy was inhibited by 3-MA. (B) Light microscopy observations at 72 h p.i. of 3-MA-treated and untreated BmN cells infected with BmNPV T3 isolate. (C) Percentage of cells displaying OBs in the nucleus. Each value is calculated at 72 h p.i. from three independent infections and for each infection, 298–360 cells were scored. Error bars indicates ±SEM. (D) Western blot analysis of polyhedrin with a mouse monoclonal anti-polyhedrin antibody.

Mentions: As described above, polyhedrin was colocalized with BmLC3 on the isolation membrane of autophagosome, prompting the question whether or not cellular autophagy played a role in polyhedra particle production. Firstly, cellular autophagy was detected during BmNPV infection. Cells were infected with the BmNPV T3 isolate, and at designated time points p.i. harvested for Western blot analysis with a rabbit polyclonal anti-BmLC3 antibody. Coomassie blue stain of total proteins was used as the internal reference in this study for normalization (Fig. 8A,D, lower panel; See also Supplementary Fig. S1). LC3 is the most widely monitored autophagy-related protein29. BmLC3 was detected as two bands (Fig. 8A). One is BmLC3-I which is cytosolic, and the other represents BmLC3-II that is present on isolation membranes of autophagosomes and pre-autophagosomes (Fig. 7A). During BmNPV infection, the level of BmLC3-I is stable. However the amount of BmLC3-II in 3-MA-treated cells is lower than that in untreated cells, suggesting that cellular autophagy was inhibited by 3-MA (Fig. 8A).


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)

Cellular autophagy played a role in polyhedra particle production.Land M, protein marker. (A) Cellular autophagy was inhibited by 3-MA. (B) Light microscopy observations at 72 h p.i. of 3-MA-treated and untreated BmN cells infected with BmNPV T3 isolate. (C) Percentage of cells displaying OBs in the nucleus. Each value is calculated at 72 h p.i. from three independent infections and for each infection, 298–360 cells were scored. Error bars indicates ±SEM. (D) Western blot analysis of polyhedrin with a mouse monoclonal anti-polyhedrin antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Cellular autophagy played a role in polyhedra particle production.Land M, protein marker. (A) Cellular autophagy was inhibited by 3-MA. (B) Light microscopy observations at 72 h p.i. of 3-MA-treated and untreated BmN cells infected with BmNPV T3 isolate. (C) Percentage of cells displaying OBs in the nucleus. Each value is calculated at 72 h p.i. from three independent infections and for each infection, 298–360 cells were scored. Error bars indicates ±SEM. (D) Western blot analysis of polyhedrin with a mouse monoclonal anti-polyhedrin antibody.
Mentions: As described above, polyhedrin was colocalized with BmLC3 on the isolation membrane of autophagosome, prompting the question whether or not cellular autophagy played a role in polyhedra particle production. Firstly, cellular autophagy was detected during BmNPV infection. Cells were infected with the BmNPV T3 isolate, and at designated time points p.i. harvested for Western blot analysis with a rabbit polyclonal anti-BmLC3 antibody. Coomassie blue stain of total proteins was used as the internal reference in this study for normalization (Fig. 8A,D, lower panel; See also Supplementary Fig. S1). LC3 is the most widely monitored autophagy-related protein29. BmLC3 was detected as two bands (Fig. 8A). One is BmLC3-I which is cytosolic, and the other represents BmLC3-II that is present on isolation membranes of autophagosomes and pre-autophagosomes (Fig. 7A). During BmNPV infection, the level of BmLC3-I is stable. However the amount of BmLC3-II in 3-MA-treated cells is lower than that in untreated cells, suggesting that cellular autophagy was inhibited by 3-MA (Fig. 8A).

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