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Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection


The mechanism of how Bombyx mori nucleopolyhedrovirus (BmNPV) enters cells is unknown. The primary components of membrane lipid rafts are proteins and cholesterol, and membrane lipid rafts are thought to be an active region for host–viral interactions. However, whether they contribute to the entry of BmNPV into silkworm cells remains unclear. In this study, we explored the membrane protein components of lipid rafts from BmN cells with mass spectrometry (MS). Proteins and cholesterol were investigated after establishing infection with BmNPV in BmN cells. In total, 222 proteins were identified in the lipid rafts, and Gene Ontology (GO) annotation analysis showed that more than 10% of these proteins had binding and catalytic functions. We then identified proteins that potentially interact between lipid rafts and BmNPV virions using the Virus Overlay Protein Blot Assay (VOPBA). A total of 65 proteins were analyzed with MS, and 7 were predicted to be binding proteins involved in BmNPV cellular invasion, including actin, kinesin light chain-like isoform X2, annexin B13, heat-shock protein 90, barrier-to-autointegration factor B-like and serine/arginine-rich splicing factor 1 A-like. When the cholesterol of the lipid rafts from the membrane was depleted by methyl-β-cyclodextrin (MβCD), BmNPV entry into BmN cells was blocked. However, supplying cholesterol into the medium rescued the BmNPV infection ability. These results show that membrane lipid rafts may be the active regions for the entry of BmNPV into cells, and the components of membrane lipid rafts may be candidate targets for improving the resistance of the silkworm to BmNPV.

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  1. Anup S, David C, Boda AR, Foster LJ, Davis MJ, Hill MM (2014) Raftprot: mammalian lipid rafts proteome database. Nucleic Acids Res, 43 (Database issue): D335–D338

  2. Bender FC, Whitbeck JC, Poncede Leon M, Lou H, Eisenberg RJ, Cohen GH (2003) Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J Virol 77: 9542–9552

  3. Blaising J, Pecheur EI (2013) Lipids: a key for hepatitis C virus entry and a potential target for antiviral strategies. Biochimie 95:96–102

  4. Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–181

  5. Burlone ME, Budkowska A (2009) Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen VirolL 90:1055–1070

  6. Chan RB, Tanner L, Wenk MR (2010) Implications for lipids during replication of enveloped viruses. Chem Phys Lipids 163:449–459

  7. Chazal N, Gerlier D (2003) Virus entry, assembly, budding, and membrane rafts. Mic & Mol. Biol Rev 67:226–237

  8. Chen HQ, Yao Q, Bao F, Chen KP, Liu XY, Li J, Wang L (2012) Comparative proteome analysis of silkworm in its susceptibility and resistance responses to Bombyx mori densonucleosis virus. Intervirology 55:21–28

  9. Clem RJ, Passarelli AL (2012) Baculoviruses: sophisticated pathogens of insects. PLoS Pathog 9:864–867

  10. Garcia RA, Pantazatos SP, Pantazatos DP, MacDonald RC (2001) Cholesterol stabilizes hemifused phospholipid bilayer vesicles. Biochim Biophys Acta 1511:264–270

  11. Hidari KI, Yamaguchi M, Ueno F, Abe T, Yoshida K, Suzuki T (2013) Influenza virus utilizes N-linked sialoglycans as receptors in A549 cells. Biochem Bioph Res Co 436: 394–402

  12. Hu XL, Shen YW, Qin Z, Wang GB, Wu XF, Gong CL (2016) Bm59 is an early gene, but is unessential for the propagation and assembly of Bombyx mori nucleopolyhedrovirus. Mol Genet Genomics 291(1):145–154

  13. Ibrahim N, Wicklund A, Jamin A, Wiebe MS (2013) Barrier to autointegration factor (BAF) inhibits vaccinia virus intermediate transcription in the absence of the viral B1 kinase. Virology 444(2):363–373

  14. Ilangumaran S, Hoessli DC (1998) Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem J 335:433–440

  15. Jacob R, Heine M, Eikemeyer J, Frerker N, Zimmer KP, Rescher U, Gerke V, Naim HY (2004) Annexin II is required for apical transport in polarized epithelial cells. J Biol Chem 279:3680–3683

  16. Janes PW, Ley SC, Magee AI, Kabouridis PS (2000) The role of lipid rafts in T cell antigen receptor (TCR) signalling. Semin Immunol 12:23–34

  17. Jitoboam K, Phaonakrop N, Libsittikul S, Thepparit C, Roytrakul S, Smith DR (2016) Actin interacts with dengue virus 2 and 4 envelope proteins. PLoS One 11(3):e0151951

  18. Jirakanwisal K, Srisutthisamphan K, Thepparit C, Suptawiwat O, Auewarakul P, Paemanee A, Roytrakul S, Smith DR (2015) Identification of Hsp90 as a species independent H5N1 avian influenza A virus PB2 interacting protein. Comp Immunol Microbiol Infect Dis 43:28–35

  19. Keller P, Simons K (1998) Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol 140:1357–1367

  20. Lorizate M and Krausslich HG (2011) Role of lipids in virus replication. Csh Perspect Biol 3: 3437–3451

  21. Manes S, del Real G, Martinez AC (2003) Pathogens: raft hijackers. Nat Rev Immunol 3:557–568

  22. Mannova P, Fang R, Wang H, Deng B, McIntosh MW, Hanash SM, Beretta L (2006) Modification of host lipid rafts proteome upon hepatitis C virus replication. Mol Cell Proteomics 5:2319–2325

  23. Martinez MA, Lopez S, Arias CF, Isa P (2013) Gangliosides have a functional role during rotavirus cell entry. J Virol 87:1115–1122

  24. Matrosovich M, Herrler G, Klenk HD (2015) Sialic acid receptors of viruses. Top Curr Chem 367:1–28

  25. Mayor S and Riezman H (2004) Sorting GPI-anchored proteins. Nat Rev Mol Cell Bio 5: 110–120.

  26. Miao XX, Xub SJ, Li MH, Li MW, Huang JH, Dai FY, Marino SW, Mills DR, Zeng P, Mita K, Jia SH, Zhang Y, Liu WB, Xiang H, Guo QH, Xu AY, Kong XY, Lin HX, Shi YZ, Lu G, Zhang X, Huang W, Yasukochi Y, Sugasaki T, Shimada T, Nagaraju J, Xiang ZH, Wang SY, Goldsmith MR, Lu C, Zhao GP, Huang YP (2005) Simple sequence repeat-based consensus linkage map of Bombyx mori. Proc Natl Acad Sci USA 102:16303–16310

  27. Morizono K, Chen IS (2014) Role of phosphatidylserine receptors in enveloped virus infection. J Virol 88:4275–4290

  28. Ono C, Kamagata T, Taka H, Sahara K, Asano S, Bando H (2012) Phenotypic grouping of 141 BmNPVs lacking viral gene sequences. Virus Res 165:197–206

  29. Perera R, Riley C, Isaac G, Hopf-Jannasch AS, Moore RJ, Weitz KW, Pasa-Tolic L, Metz TO, Adamec J, Kuhn RJ (2012) Dengue virus infection perturbs lipid homeostasis in infected mosquito cells. PLoS Pathog 8: e1002584–e1002584

  30. Pike LJ (2003) Lipid rafts: bringing order to chaos. J Lipid Res 44(4):655–667

  31. Razinkov VI, Cohen FS (2000) Sterols and sphingolipids strongly affect the growth of fusion pores induced by the hemagglutinin of influenza virus. BioChemistry 39:13462–13469

  32. Sagan SM, Rouleau Y, Leggiadro C, Supekova L, Schultz PG, Su AI, Pezacki JP (2006) The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication. Biochem Cell Biol 84:67–79

  33. Schechtman D, Murriel C, Bright R, Mochly-Rosen D (2003) Overlay method for detecting protein–protein interactions. Methods Mol Biol 233:351–357

  34. Schneider-Schaulies J, Schneider-Schaulies S (2013) Viral infections and sphingolipids. Handb Exp Pharmacol 216:321–340

  35. Shi ST, Lee KJ, Aizaki H, Hwang SB, Lai MM (2003) Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2. J Virol 77: 4160–4167

  36. Slack J, Arif BM (2007) The baculoviruses occlusion-derived virus: virion structure and function. Adv Virus Res 69:99–165

  37. Stiasny K, Koessl C, Heinz FX (2003) Involvement of lipids in different steps of the flavivirus fusion mechanism. J Virol 77:7856–7862

  38. Waheed AA, Freed EO (2010) The role of lipids in retrovirus replication. Viruses 2:1146–1180

  39. Workenhe ST, Ketela T, Moffat J, Cuddington BP, Mossman KL (2016) Genome-wide lentiviral shRNA screen identifies serine/arginine-rich splicing factor 2 as a determinant of oncolytic virus activity in breast cancer cells. Oncogene 35:2465–2474

  40. Xiang XW, Shen YW, Yang R, Chen L, Hu XL, Wu XF (2013) Bombyx mori nucleopolyhedrovirus BmP95 plays an essential role in budded virus production and nucleocapsid assembly. J Gen Virol 94:1669–1679

  41. Xue J, Qiao N, Zhang W, Cheng RL, Zhang XQ, Bao YY, Xu YP, Gu LZ, Han JD, Zhang CX (2012) Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. J Virol 86:7345–7359

  42. Yang Z, Shi ZX, Guo HC, Qu H, Zhang Y, Tu CC (2015) Annexin 2 is a host protein binding to classical swine fever virus E2 glycoprotein and promoting viral growth in PK-15 cells. Virus Res 201:16–23

  43. Ye J, Fang L, Zheng H, Zhang Y, Chen J, Zhang Z, Wang J, Li S, Li R, Bolund L, Wang J (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34:293–299

  44. Zdobnov EM, Apweiler R (2001) InterProScan–an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–854

  45. Zhang J, Pekosz A, Lamb RA (2000) Influenza virus assembly and lipid rafts microdomains: a role for the cytoplasmic tails of the spike glycoproteins. J Virol 74:4634–4644

  46. Zhou Y, Gao L, Shi H, Xia H, Gao L, Lian C, Chen L, Yao Q, Chen K, Liu X (2013) Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus. Genomics 101:256–262

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Correspondence to Chengliang Gong.

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The authors declare that they have no conflicts of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.


This study was funded by the Natural Science Foundation of Jiangsu Higher Education Institutions of China (15KJB230002), the Natural Science Foundation of Jiangsu Province (BK20140324), the National Natural Science Foundation of China (31072085) and a project funded by the Priority Academic Program of Development of Jiangsu Higher Education Institutions.

Additional information

X. Hu and M. Zhu contributed equally to this work.

Communicated by Q. Xia.

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Hu, X., Zhu, M., Liang, Z. et al. Proteomic analysis of BmN cell lipid rafts reveals roles in Bombyx mori nucleopolyhedrovirus infection. Mol Genet Genomics 292, 465–474 (2017). https://doi.org/10.1007/s00438-016-1284-y

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  • Bombyx mori
  • Bombyx mori nucleopolyhedrovirus
  • Cholesterol
  • Lipid rafts