Abstract
Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.
Highlights
The silkworm (B. mori) has long been raised as a beneficial insect in sericulture industry and a model insect of Lepidoptera in research
Our preliminary proteomic analysis showed the expression of VATPase was up-regulated in B. mori nucleopolyhedrovirus (BmNPV)-resistant strain NB, indicating that V-ATPase may play a role in the silkworm defense response against BmNPV
The numbers of obtained occlusion bodies (OBs) and cells were examined using a hemocytometer under light microscope, and the titers of budded viruses (BVs) were determined by a tissue culture infectious dose 50 (TCID50) method based on endpoint dilution [25]
Summary
The silkworm (B. mori) has long been raised as a beneficial insect in sericulture industry and a model insect of Lepidoptera in research. Certain silkworm metabolites and proteins have been shown to possess antiviral activity against infection of BmNPV [3,4,5,6], but the mechanism of B. mori defense response against BmNPV remains largely unknown. V-ATPases locate in various endomembrane systems and plasma membranes [11,12] They are multi-subunit complexes organized into a peripheral domain (V1) responsible for ATP hydrolysis and an integral domain (V0) that transports protons across membranes [13,14,15], and are essential for pH regulation of the intracellular compartments, the cytoplasm and the extracellular space [16,17,18]. Our preliminary proteomic analysis showed the expression of VATPase was up-regulated in BmNPV-resistant strain NB (unpublished data), indicating that V-ATPase may play a role in the silkworm defense response against BmNPV
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