Identification Of Bombyx Mori Receptor Expression-enhancing Protein And Its Function In The BmNPV Invasion

Baculovirus is a class of envelope virus with 80-180 kb double-stranded circular DNA genomes, which specificly chooses arthropods as their host. They are widely use d as insect pesticide, gene therapy vector and eukaryotic expression vector as they can not infect vertebrates and plants. Baculovirus, which belongs to Baculoviridae, can be divided into Alpha-Baculovirus, Beta-Baculovirus, Gamma-Baculovirus and Delta-Baculo virus according to their hosts and be classified into GV and NPV according to their Morphological characteristics. Bombyx mori nucleopolyhedrovirus(Bm NPV) is a typical kind of NPV, which is a trigger of nucleopolyhedrosis(sepsis disease) that leaded to almost 70% of economic losses caused by silkworm disease, that affected the yield an d quality of silk seriously, that threatened the stability of silk industry and its domina nce in the international market. Recently, increasing studies focused on Bm NPV were performed, the mechanism of its invasion, however, was still unclear. In our laboratory,we kept two silkworm cell lines, Bm N-SWU1 and Bm N-SWU2, which established fr om the same ovarian tissue but showed very different susceptibility to Bm NPV infecti on―Bm N-SWU1 cells were sensitive to Bm NPV infection while Bm N-SWU2 cells we re resistant to Bm NPV infection. Our previous studies found that the Bm NPV invasion in Bm N-SWU2 cells was limited. In this study, we try to explore the main reason o f this limitation through the proteomic analysis between Bm N-SWU1 and Bm N-SWU2 cells. The main results are as follows: 1. Differential proteomic analysis of Bm N-SWU1 and Bm N-SWU2 cellsTo investigate the mechanism of the limitation during the process of Bm NPV inv asion in Bm N-SWU2 cells, we performed i TRAQ-based differential proteomic analysis using these two cell lines. A total of 629 proteins were differentially expressed between the Bm N-SWU2 and Bm N-SWU1 cells with 348 up-regulated and 281 down-regulat ed proteins. Further analysis identified only four differentially expressed predicted membra ne proteins. Notably, BGIBMGA013777 was the only membrane protein showed more than5-fold differences in expression and can enhance the function of membrane receptor s. The result of q RT-PCR showed that BGIBMGA013777 expression level in Bm N-SWU1 w as significantly higher than that in Bm N-SWU2 cells. Finally, we chose this protein as t he target protein. 2. The characterazation and localization of Bm REEPaReceptor expression-enhancing protein(REEP) family is a family that can enhance receptor function and highly conserve across different species. REEPs contain a TB2/DP1, HVA22 domain which involved in the cellular transport and secretion.This family consists of six members namely REEP1-REEP6 respectively and can be divided into two subfamilies REEP1-REEP4 and REEP5-REEP6. There are generally 5 to 6 REEP genes in vertebrates, and two REEP genes in invertebrates. Sequence analysis showed t hat Bm REEPa had a TB2/DP1, HVA22 domain, which is conserved in the REEP protein fa mily, as well as three transmembrane domains. Phylogenetic analysis showed that the ded uced amino acid sequence of Bm REEPa clustered with the REEP5-REEP6 subfamily. The results of fluorescence and western blotting analysis indicated that Bm REEPa is l ocalized in the cell membrane with its C-terminus in the cytoplasm and the N-terminu s outside the cell. 3. The influences of Bm REEPa during Bm NPV invasionPrevious study showed that the entry of Bm NPV into Bm N-SWU2 cells were lim ated and the limitation was presented on cell membrane. Our previous study in this re search identified a membrane protein, Bm REEPa, was differentially expressed in Bm NSWU1 and Bm N-SWU2 cells, which related to cell surface-receptor function. In this p art, we investigated the function of Bm REEPa in Bm NPV invasion by gene interferenc e in Bm N-SWU1 cells and over-expression in Bm N-SWU2 cells. The results showed t hat the introduction of Bm NPV in Bm N-SWU1, which is sensitive to Bm NPV infectio n, was inhibited, while the limitation of Bm NPV invasion in Bm N-SWU2, which is re sistant to Bm NPV infection, was rescued to a certain extent. Furthermore, we found t hat Bm REEPa can interact with GP64 which is the essential envelope protein for Bm NPV invasion. In addition, we get Bm REEPa interfered silkworm strain by using microinjection technology, and the results indicated that the infection ability of transgenic s train was obviously weaker than normal strain. We also found that the inhibition of B m NPV infection was mainly functioned in secondary infection which is the infection o f BV particles. 4. Identification of Bm REEPa interacting proteinIn this part, we screened Bm REEPa interacting proteins through Co-IP by using Bm REEPa as a bait protein, and identified a receptor protein, Bm Ptchd, which related to cholesterol transport. Further study showed that Bm Ptchd can form a complex with Bm REEPa and GP64. Additionally, our study indicated that the expression level of B m REEPa and Bm Ptchd was up-regulated when cells treated with MβCD, and the expr ession level of Bm Ptchd was down-regulated in cholesterol content increased cells, the expression level of Bm REEPa, however, was still up-regulated. Thus, we speculated th at Bm REEPa can participant not only cholesterol transport and metabolism but also ch olesterol synthetizing, while Bm Ptchd can only functioned in cholesterol transporting.From this study, we know that Bm REEPa localize in the cell membrane with its C-terminus in the cytoplasm and N-terminus outside the cell. Bm REEPa can affect Bm NPV invasion both in cells and larvae, especially during BV infection. In addition, we find that Bm REEPa is important for cholesterol regulation, and cholesterol is essential for Bm NPV entry. In summary, we believe Bm REEPa can play an important role in Bm NPV invasion. Our results will help us explain the detailed mechanism of Bm NPV infection and give a new insight for REEPs research.