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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

The polyhedrin was targeted to aggresome.(A) Colocalization of polyhedrin-mCherry and the heterogenous aggresomal marker GFP-250, and of coalesced aggregates of both GFP-250 and polyhedrin-mCherry with γ−tubulin. Plasmids pPie1-GFP-250 and pPph-Polyhedrin-mCherry were employed for transposition and transfection. Equal MOI (10 TCID50/cell) of P2 viral stocks individually expressing GFP-250 and polyhedrin-mCherry were used to co-infect BmN cells and then analyzed at 24 h p.i. by a confocal fluorescence microscopy, and in parallel, by immunostaining for γ−tubulin. In merged images, yellow dots suggested colocalization between GFP-250 and polyhedrin-mCherry (Merge I), and white ones indicated colocalization of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). (B) Percentage of cells showing colocalization of GFP-250 with polyhedrin-mCherry (Merge I), and of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). Each value is summarized from three independent infections. For each infection, 174–190 cells were scored for complete and partial colocalization data collection. Error bars indicates ±SEM.
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f4: The polyhedrin was targeted to aggresome.(A) Colocalization of polyhedrin-mCherry and the heterogenous aggresomal marker GFP-250, and of coalesced aggregates of both GFP-250 and polyhedrin-mCherry with γ−tubulin. Plasmids pPie1-GFP-250 and pPph-Polyhedrin-mCherry were employed for transposition and transfection. Equal MOI (10 TCID50/cell) of P2 viral stocks individually expressing GFP-250 and polyhedrin-mCherry were used to co-infect BmN cells and then analyzed at 24 h p.i. by a confocal fluorescence microscopy, and in parallel, by immunostaining for γ−tubulin. In merged images, yellow dots suggested colocalization between GFP-250 and polyhedrin-mCherry (Merge I), and white ones indicated colocalization of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). (B) Percentage of cells showing colocalization of GFP-250 with polyhedrin-mCherry (Merge I), and of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). Each value is summarized from three independent infections. For each infection, 174–190 cells were scored for complete and partial colocalization data collection. Error bars indicates ±SEM.

Mentions: As an efficient way for cell to avoid proteotoxicity, aggresome is formed to sequester and inactivate these potentially harmful aggregated proteins. These findings above raise the possibility that cytoplasmic foci could represent targeting of polyhedrin proteins to aggresomes. Thus to confirm whether or not some polyhedrin aggregates are aggresomes, the heterogenous aggresomal marker GFP-250, together with the fusion protein polyhedrin-mCherry, was expressed in BmN cells. Aggresomes formed by polyhedrin were portrayed by colocalization of chimera GFP-250 with polyhedrin-mCherry. GFP-250 consists of eGFP fused at its C-terminus to the first 250 amino acids of p115, and was shown to result in a spherical aggresome in previous study24. At 24 h p.i., co-infected cells contained aggregates of both GFP-250 and polyhedrin-mCherry in the cytoplasm. Moreover, there was a high degree of colocalization between these aggregates, as evidenced by complete and partial colocalization of GFP-250 with polyhedrin-mCherry in 80.5 ± 3.9% of BmN cells (Fig. 4A,B).


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)

The polyhedrin was targeted to aggresome.(A) Colocalization of polyhedrin-mCherry and the heterogenous aggresomal marker GFP-250, and of coalesced aggregates of both GFP-250 and polyhedrin-mCherry with γ−tubulin. Plasmids pPie1-GFP-250 and pPph-Polyhedrin-mCherry were employed for transposition and transfection. Equal MOI (10 TCID50/cell) of P2 viral stocks individually expressing GFP-250 and polyhedrin-mCherry were used to co-infect BmN cells and then analyzed at 24 h p.i. by a confocal fluorescence microscopy, and in parallel, by immunostaining for γ−tubulin. In merged images, yellow dots suggested colocalization between GFP-250 and polyhedrin-mCherry (Merge I), and white ones indicated colocalization of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). (B) Percentage of cells showing colocalization of GFP-250 with polyhedrin-mCherry (Merge I), and of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). Each value is summarized from three independent infections. For each infection, 174–190 cells were scored for complete and partial colocalization data collection. Error bars indicates ±SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The polyhedrin was targeted to aggresome.(A) Colocalization of polyhedrin-mCherry and the heterogenous aggresomal marker GFP-250, and of coalesced aggregates of both GFP-250 and polyhedrin-mCherry with γ−tubulin. Plasmids pPie1-GFP-250 and pPph-Polyhedrin-mCherry were employed for transposition and transfection. Equal MOI (10 TCID50/cell) of P2 viral stocks individually expressing GFP-250 and polyhedrin-mCherry were used to co-infect BmN cells and then analyzed at 24 h p.i. by a confocal fluorescence microscopy, and in parallel, by immunostaining for γ−tubulin. In merged images, yellow dots suggested colocalization between GFP-250 and polyhedrin-mCherry (Merge I), and white ones indicated colocalization of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). (B) Percentage of cells showing colocalization of GFP-250 with polyhedrin-mCherry (Merge I), and of coalesced GFP-250/polyhedrin-mCherry aggregate with γ−tubulin (Merge II). Each value is summarized from three independent infections. For each infection, 174–190 cells were scored for complete and partial colocalization data collection. Error bars indicates ±SEM.
Mentions: As an efficient way for cell to avoid proteotoxicity, aggresome is formed to sequester and inactivate these potentially harmful aggregated proteins. These findings above raise the possibility that cytoplasmic foci could represent targeting of polyhedrin proteins to aggresomes. Thus to confirm whether or not some polyhedrin aggregates are aggresomes, the heterogenous aggresomal marker GFP-250, together with the fusion protein polyhedrin-mCherry, was expressed in BmN cells. Aggresomes formed by polyhedrin were portrayed by colocalization of chimera GFP-250 with polyhedrin-mCherry. GFP-250 consists of eGFP fused at its C-terminus to the first 250 amino acids of p115, and was shown to result in a spherical aggresome in previous study24. At 24 h p.i., co-infected cells contained aggregates of both GFP-250 and polyhedrin-mCherry in the cytoplasm. Moreover, there was a high degree of colocalization between these aggregates, as evidenced by complete and partial colocalization of GFP-250 with polyhedrin-mCherry in 80.5 ± 3.9% of BmN cells (Fig. 4A,B).

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