Dong Xiao-long, Liu Tai-hang, Wang Wei, Pan Cai-xia, Wu Yun-fei, Du Guo-yu, Chen Peng, Lu Cheng, Pan Min-hui
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.
Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing, China.
PLoS One. 2015 Dec 14;10(12):e0144575. doi: 10.1371/journal.pone.0144575. eCollection 2015.
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.
我们之前从家蚕卵巢中建立了两个家蚕细胞系，即BmN-SWU1和BmN-SWU2。BmN-SWU1细胞对家蚕核型多角体病毒（BmNPV）感染敏感，而BmN-SWU2细胞对BmNPV感染具有高度抗性。有趣的是，我们发现BmNPV进入BmN-SWU2细胞的过程在很大程度上受到抑制。为了探究这种抑制机制，在本研究中，我们使用基于等压标签相对和绝对定量（iTRAQ）的定量蛋白质表达谱分析，鉴定出这两个细胞系之间有629种差异表达蛋白。其中，我们鉴定出一种新的膜蛋白，命名为BmREEPa。编码BmREEPa的基因转录出两种剪接变体；一种长573 bp的BmREEPa-L，编码一个含190个氨基酸的蛋白质，另一种长501 bp的BmREEPa-S，编码一个含166个氨基酸的蛋白质。BmREEPa包含一个保守的TB2/DP、HVA22结构域和三个跨膜结构域。它定位于质膜，C端在细胞质中，N端在细胞外。我们发现，限制BmN-SWU1细胞中BmREEPa的表达会抑制BmNPV的进入，而在BmN-SWU2细胞中过表达BmREEPa则会促进BmNPV的进入。我们的结果还表明，BmREEPa可以与GP64相互作用，GP64是BmNPV进入细胞的关键包膜融合蛋白。综上所述，我们的研究结果表明，BmREEPa是BmNPV进入家蚕细胞所必需的，可能为鉴定BmNPV受体提供思路。
