Abstract
We previously established two silkworm cell lines, BmN-SWU1 and BmN-SWU2, from Bombyx mori ovaries. BmN-SWU1 cells are susceptible while BmN-SWU2 cells are highly resistant to BmNPV infection. Interestingly, we found that the entry of BmNPV into BmN-SWU2 cells was largely inhibited. To explore the mechanism of this inhibition, in this study we used isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative protein expression profiling and identified 629 differentially expressed proteins between the two cell lines. Among them, we identified a new membrane protein termed BmREEPa. The gene encoding BmREEPa transcribes two splice variants; a 573 bp long BmREEPa-L encoding a protein with 190 amino acids and a 501 bp long BmREEPa-S encoding a protein with 166 amino acids. BmREEPa contains a conserved TB2/DP, HVA22 domain and three transmembrane domains. It is localized in the plasma membrane with a cytoplasmic C-terminus and an extracellular N-terminus. We found that limiting the expression of BmREEPa in BmN-SWU1 cells inhibited BmNPV entry, whereas over-expression of BmREEPa in BmN-SWU2 cells promoted BmNPV entry. Our results also indicated that BmREEPa can interact with GP64, which is the key envelope fusion protein for BmNPV entry. Taken together, the findings of our study revealed that BmREEPa is required for BmNPV to gain entry into silkworm cells, and may provide insights for the identification of BmNPV receptors.
Highlights
The baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen of silkworm, which is an economically important insect and lepidopteran model
We characterized the BmREEPa gene and identified the following properties in its putative protein; sequence analysis revealed that BmREEPa contains a highly conserved domain characteristic of the Receptor expression-enhancing protein (REEP) family (TB2/ DP1, HVA22) and three transmembrane domains; phylogenetic analysis demonstrated that BmREEPa belongs to the REEP5-REEP6 subfamily; BmREEPa was localized on the cell membrane and exhibited a unique topology with the N-terminus being extracellular and the C-terminus being cytoplasmic
We found that after BmNPV infection, BmREEPadepleted BmN-SWU1 cells had significantly lesser number of virus-infected cells and viral particles, and lower VP39 expression level in comparison to the control BmN-SWU1 cells
Summary
The baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen of silkworm, which is an economically important insect and lepidopteran model. Its infection leads to about 70% annual loss in sericulture [1,2]. Because the mechanism of BmNPV resistance in silkworm is unclear, silkworm strains with high resistance to BmNPV infections have not been established. The mechanism underlying BmNPV resistance in silkworm has become a subject of intensive investigation. Previous studies attempted to explore this resistance. PLOS ONE | DOI:10.1371/journal.pone.0144575 December 14, 2015
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