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

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.

Protein composition analysis of polyhedra matrix of Bombyx mori nucleopolyhedrovirus (BmNPV) showed powerful capacity of polyhedra to encapsulate foreign proteins

“…The presence of dodecameric or disulfide-linked octameric polyhedrin molecules in the polyhedra has been reported 167 , 168 . Recently, polyhedrin was found to form aggregates and aggresomes in the cytoplasm of infected cells 166 , 169 . Polyhedrin directly binds to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and is co-localized with BmLC3 to the isolation membrane of the autophagosome 166 .…”

“…Recently, polyhedrin was found to form aggregates and aggresomes in the cytoplasm of infected cells 166 , 169 . Polyhedrin directly binds to Bombyx mori microtubule-associated protein 1-light chain 3 (BmLC3), an autophagosome marker, and is co-localized with BmLC3 to the isolation membrane of the autophagosome 166 . These findings suggest that polyhedrin may function as an autophagic adapter during the process of selective autophagy, which is regulated by post-translational modifications, such as phosphorylation and ubiquitination 170 , 171 .…”

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Figure 5 Western blot analysis of the protein polyhedrin in the polyhedra matrix. Total proteins of the polyhedra matrix fraction were subjected to SDS-PAGE and then Western blot using a mouse monoclonal anti-polyhedrin antibody 166 . Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a chemiluminescence detection system.

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BmNPV infection correlates with the enhancement of the resistance of Bombyx mori cells to UV radiation

“…Recombinant BmNPV (vBm Pie‐1‐egfp ) expressing enhanced green fluorescent protein (EGFP) was constructed in previous work (Tang et al, ). Plasmids pHTB‐P ie‐1 ‐mCherry expressing the mCherry reporter gene were constructed by Guo et al () in our laboratory. BmN cells were cultured at 27°C in TC‐100 insect medium supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA).…”

Identification and Characterization of BmVta1, a Bombyx mori (Lepidoptera: Bombycidae) Homologue for Vta1 That is Up-Regulated in Development

“…The BmN cells were cultured in TC100 medium supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin at 27 °C. The recombinant plasmids pIB-EGFP, pIB-EGFP-BmVta1, and pIB-BmVps4-EGFP were, respectively, transfected into BmN cells by liposome method as reported previously ( Guo et al 2015 , Lu et al 2017 ). Briefly, 8 μl cellfectin II reagent was diluted into 100 μl TC100 medium, mixed thoroughly and incubated for 30 min at room temperature, then was mixed with 100 μl TC100 medium containing 3 μg of plasmid and incubated for another 30 min to form liposomes.…”