Characterization Of Aggregate/aggresome Structures Formed During Infection Of Bombyx Mori Nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus(BmNPV) infection leads to formation of aggregates/aggresomes in BmN cells. Aggregates/aggresomes, toxic to host cells, play an important role in virus infection. Baculovirus are nucleus DNA pathogens infecting insects of the orders Lepidoptera, Hymenoptera, and Diptera, however, the components and functions of aggregates/aggresomes induced by baculovirus are still unknown. In this study, we will explore the toxicological effects of BmNPV proteins on BmN cells,and host cell reactions to viral proteotoxic stress, providing more information for replication mechanism of this virus.1. A large number of aggregates/aggresomes formed during BmNPV infectionFirst, production of aggregates and aggresomes was detected during infection of BmNPV with a fluorescent molecular dye. Furthermore, GP64 and polyhedrin forming aggregates/aggresomes was confirmed by filter trap assay. The aggregates/aggresomes were co-localized with some viral proteins such as GP64, Bm56, Bm75 and Bm101.Some very late expressed products, for instance polyhedrin, also formed aggregates and aggresomes in the cytoplasm, and then the number of these structures decreased accompanying progress of BmNPV infection, implying the versatile roles of aggregates/aggresomes at different stages during BmNPV infection. Finally, the stretches with high fibrillation propensities were predicted to be contained in amino acid sequences of GP64 and polyhedrin by a 3D profile method. The results indicated that some viral proteins become aggregates/aggresomes during BmNPV infection.2. Effects of host HSC/HSP70 molecular chaperone systemAggregates/aggresomes were co-localized with heat shock cognates/proteins of the70-kDa family(HSC/HSP70s), ubiquitinated proteins and recruited the mitochondria.At 24 h and 48 h p.i., using immunofluorescence microscopy we found that traffic of viral protein Bm56 and polyhedrin into the nuclei was inhibited by quercetin, an inhibitor of HSC/HSP70, indicating that HSC/HSP70 chaperone system plays a role inproduction and trafficking of some viral proteins.3. Some viral proteins formed aggresomesNext, whether some viral proteins formed aggresomes was confirmed using several recombinant viruses(i.e. BmBac-GFP250, BmBac-GP64-mCherry and BmBac-mCherry-Bm101) expressing different fluorescent labels. These viruses co-infected BmN cells. GP64 and Bm101 were co-localized with the aggresomal marker GFP250, suggesting that some viral proteins formed aggresomes.4. Effects of cellular autophagy on BmNPV propagationThe cellular microtubule-associated protein 1-light chain 3(LC3), the autophagosome marker, was cloned to elucidate the role of celluar autophagy on BmNPV progeny propagation. It was observed that autophagy was induced by BmNPV infection. Some viral proteins were co-localized with LC3, indicating that these proteins might play a role in virus-associated autophagosome formation, or were degraded by cellular autophagy. The production of polyhedrin and number of polyhedra particles were significantly reduced in the presence of 3-methyladenine(3-MA), an inhibitor of cellular autophagy. These results indicated that BmNPV induced autophagy to promote virus replication.