BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells

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.

Fig 1. Proteins differently expressed between BmN-SWU1 and BmN-SWU2 via proteomic analysis.

(A) GO categories of differently expressed proteins between BmN-SWU1 and BmN-SWU2. A ratio of BmN-SWU2/BmN-SWU1 ≥ 1.5 was considered as up-regulated (brown bar) and ≤ 0.66 was considered down-regulated (purple bar); (B) classification of KEGG pathways associated with the differentially expressed proteins between BmN-SWU1 and BmN-SWU2. The ratio of BmN-SWU2/BmN-SWU1 ≥1.5 was considered up-regulated (brown bar) and ≤ 0.66 was considered down-regulated (blue bar).

Fig 2. Two different splicesomes of BmREEPa.

(A) Gene structure of two BmREEPa splicesomes. The red region is lacking in BmREEPa-S. (B) Differences between the gene sequences of the two BmREEPa splicesomes; the sequence in red is not present in BmREEPa-S; black underline represents the start and stop codons.

Fig 3. Homology analysis of BmREEPa.

(A) Sequence analysis of BmREEPa. Black color represents the TB2/DP1, HVA22 domain; red represents transmembrane domain; (B) phylogenetic tree (N-J) of BmREEPa with REEP genes from vertebrates and invertebrates.

Fig 4. BmREEPa localization and topology model.

(A) BmREEPa full-length, N-terminal and C-terminal sequence localization; (B) Western blot of full-length BmREEPa in the isolated membrane and cytosol proteins; (C) Western blot of BmN-SWU1 cells and culture fluid expressing BmREEPa N- and C-termini; (D) Topology model of BmREEPa. BmREEPa-L, BmREEPa-S, BmREEPa-N terminus and BmREEPa-C terminus were co-expressed with Flag tags and DsRed. DsRed was used as a marker in fluorescence observation while anti-Flag antibody was used in Western blots.

Fig 5. Effect of BmNPV transduction in BmREEPa-RNAi BmN-SWU1 cells.

(A) Green fluorescence in cells at 48 h post-infection with v39K^prm-eGFP BVs; (B) Analysis of VP39 expression in BmN-SWU1 48 h after infection with v39K^prm-eGFP BVs; (C) Viral titer in cell culture fluid 48 h after infection with v39K^prm-eGFP BVs. BmN-SWU1 without v39K^prm-eGFP BVs, BmN-SWU1 with v39K^prm-eGFP BVs and DsRed-RNAi with v39K^prm-eGFP BVs were used as controls; * indicates significant differences at P < 0.05, ** indicates significant differences at P < 0.01 with respect to the control.

Fig 6. Effect of BmNPV transduction in BmN-SWU2 cells over-expressing BmREEPa.

(A) Green fluorescence in cells infected with v39K^prm-eGFP BVs; (B) Analysis of VP39 expression in cells 48 h after infection with v39K^prm-eGFP BVs; (C) Viral titer in cell culture fluid at 48 h post-infection with v39K^prm-eGFP BVs. BmN-SWU2 without v39K^prm-eGFP BVs, BmN-SWU2 with v39K^prm-eGFP BVs and with v39K^prm-eGFP BVs were used as controls; * indicates significant differences at P < 0.05, ** indicates significant differences at P < 0.01 when compared to the control.

Fig 7. Viral titer at different stages of infection.

(A) Viral titer in BmREEPa-RNAi BmN-SWU1 and control BmN-SWU1 cell culture fluid at 0 h, 6 h, 24 h, 48 h and 96 h after infection with v39K^prm-eGFP BVs; (B) Viral titer in BmREEPa over-expressing BmN-SWU2 and control BmN-SWU2 cell culture fluid at 0 h, 6 h, 24 h, 48 h and 96 h after infection with v39K^prm-eGFP BVs. BmN-SWU1 and BmN-SWU2 without v39K^prm-eGFP BVs, BmN-SWU1 and BmN-SWU2 with v39K^prm-eGFP BVs were used as controls.

Fig 8. Function of BmREEPa during BmNPV entry.

(A) Green fluorescence in BmN-SWU2 infected with vHSP70^prm-eGFP BVs, BmN-SWU2 without vHSP70^prm–eGFP BVs, BmN-SWU2 with vHSP70^prm-eGFP BVs and pIZ-V5/his with vHSP70^prm-eGFP BVs were used as controls; (B) Western blot analysis determining the interaction between BmREEPa and GP64; P indicates lysate, S supernatant, P pellet; IgG was used as the control.