Skip to main content

Advertisement

SpringerLink
Log in

An efficient method for multigene co-interference by recombinant Bombyx mori nucleopolyhedrovirus

  • Original Article
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Bombyx mori Nucleopolyhedrovirus (BmNPV), which is a member of the Baculoviridae family, is a significant pathogen of the silkworm. The infection of BmNPV is often lethal and causes about 20% loss of cocoon in the silk industry annually. To explore the effects of different gene inhibition strategies on the replication cycle of baculovirus, we constructed the mutant virus to infect BmN cells directly and further identified ie0, ie1, and gp64 as the essential viral genes of BmNPV. To elucidate the significance of the inhibition effect of different interference strategies, we characterized and constructed the recombinant BmNPV that carried a single or multigene-interfering cassette. The results showed that the inhibition effect of dsie1 on target gene expression, virus titer, and silkworm mortality was significantly better than that of dsie0 and dsgp64. It also showed that the dsie1 interference produced fewer progeny virions and was less lethal, which indicates that ie1 played a more critical role in the BmNPV replication cycle. Furthermore, the inhibitory effect of the virus titer and mortality indicated that the multigene co-interference constructed by the baculovirus expression system was significantly better than the interference of any single-gene (p < 0.05). In summary, the strategy of multigene synergy can achieve the function of continuous interference and provide a new platform for the breeding of silkworm disease resistant. In addition, this strategy improves the various traits of the silkworm.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818):363–366

    Article  CAS  PubMed  Google Scholar 

  • Blissard GW, Wenz JR (1992) Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66(11):6829–6835

    CAS  PubMed  PubMed Central  Google Scholar 

  • Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567):550–553

    Article  CAS  PubMed  Google Scholar 

  • Carbonell LF, Klowden MJ, Miller LK (1985) Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J Virol 56(1):153–160

    CAS  PubMed  PubMed Central  Google Scholar 

  • Chen S, Hou C, Bi H, Wang Y, Xu J, Li M, James AA, Huang Y, Tan A (2017) Transgenic clustered regularly interspaced short palindromic repeat/Cas9-mediated viral gene targeting for antiviral therapy of Bombyx mori nucleopolyhedrovirus. J Virol 91(8):e02465–e02416. (https://doi.org/10.1128/JVI.02465-16)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng D, Xia Q, Duan J, Wei L, Huang C, Li Z, Wang G, Xiang Z (2008) Nuclear receptors in Bombyx mori: insights into genomic structure and developmental expression. Insect Biochem Mol Biol 38(12):1130–1137

    Article  CAS  PubMed  Google Scholar 

  • Chi JT, Chang HY, Wang NN, Chang DS, Dunphy N, Brown PO (2003) Genomewide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci USA 100(11):6343–6346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi J, Guarino LA (1995) The baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect cell factors. J Virol 69(7):4548–4551

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dai X, Willis LG, Huijskens I, Palli SR, Theilmann DA (2004) The acidic activation domains of the baculovirus transactivators IE1 and IE0 are functional for transcriptional activation in both insect and mammalian cells. J Gen Virol 85(Pt 3):573–582

    Article  CAS  PubMed  Google Scholar 

  • de Jong J, Theilmann DA, Arif BM, Krell PJ (2011) Immediate-early protein ME53 forms foci and colocalizes with GP64 and the major capsid protein VP39 at the cell membranes of Autographa californica multiple nucleopolyhedrovirus-infected cells. J Virol 85(19):9696–9707

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Dougherty WG, Parks TD (1995) Transgenes and gene suppression: telling us something new? Curr Opin Cell Biol 7(3):399–405

    Article  CAS  PubMed  Google Scholar 

  • Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001a) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836):494–498

    Article  CAS  PubMed  Google Scholar 

  • Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T (2001b) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 20(23):6877–6888

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811

    Article  CAS  PubMed  Google Scholar 

  • Gomi S, Zhou CE, Yih W, Majima K, Maeda S (1997) Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230(1):35–47

    Article  CAS  PubMed  Google Scholar 

  • Gomi S, Majima K, Maeda S (1999) Sequence analysis of the genome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80(Pt 5):1323–1337

    Article  CAS  PubMed  Google Scholar 

  • Guarino LA, Summers MD (1986) Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene. J Virol 57(2):563–571

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hammond SM, Caudy AA, Hannon GJ (2001) Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2(2):110–119

    Article  CAS  PubMed  Google Scholar 

  • Hannon GJ (2002) RNA interference. Nature 418(6894):244–251

    Article  CAS  PubMed  Google Scholar 

  • Isobe R, Kojima K, Matsuyama T, Quan GX, Kanda T, Tamura T, Sahara K, Asano SI, Bando H (2004) Use of RNAi technology to confer enhanced resistance to BmNPV on transgenic silkworms. Arch Virol 149(10):1931–1940

    CAS  PubMed  Google Scholar 

  • Jiang L, Xia Q (2014) The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. Insect Biochem Mol Biol 48:1–7

    Article  CAS  PubMed  Google Scholar 

  • Jiang L, Wang G, Cheng T, Yang Q, Jin S, Lu G, Wu F, Xiao Y, Xu H, Xia Q (2012) Resistance to Bombyx mori nucleopolyhedrovirus via overexpression of an endogenous antiviral gene in transgenic silkworms. Arch Virol 157(7):1323–1328

    Article  CAS  PubMed  Google Scholar 

  • Jiang L, Zhao P, Wang G, Cheng T, Yang Q, Jin S, Lin P, Xiao Y, Sun Q, Xia Q (2013) Comparison of factors that may affect the inhibitory efficacy of transgenic RNAi targeting of baculoviral genes in silkworm, Bombyx mori. Antiviral Res 97(3):255–263

    Article  CAS  PubMed  Google Scholar 

  • Kadlec J, Loureiro S, Abrescia NG, Stuart DI, Jones IM (2008) The postfusion structure of baculovirus gp64 supports a unified view of viral fusion machines. Nat Struct Mol Biol 15(10):1024–1030

    Article  CAS  PubMed  Google Scholar 

  • Kovacs GR, Choi J, Guarino LA, Summers MD (1992) Functional dissection of the Autographa californica nuclear polyhedrosis virus immediate-early 1 transcriptional regulatory protein. J Virol 66(12):7429–7437

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kuzio J, Rohel DZ, Curry CJ, Krebs A, Carstens EB, Faulkner P (1984) Nucleotide sequence of the p10 polypeptide gene of Autographa californica nuclear polyhedrosis virus. Virology 139(2):414–418

    Article  CAS  PubMed  Google Scholar 

  • Leonard JN, Schaffer DV (2005) Computational design of antiviral RNA interference strategies that resist human immunodeficiency virus escape. J Virol 79(3):1645–1654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li G, Tang Q, Chen H, Yao Q, Ning D, Chen K (2011) Display of Bombyx mori nucleopolyhedrovirus GP64 on the Bacillus subtilis spore coat. Curr Microbiol 62(5):1368–1373

    Article  CAS  PubMed  Google Scholar 

  • Liu Y, Zhang L, Zhang Y, Liu D, Du E, Yang Z (2015) Functional analysis of RNAi suppressor P19 on improving baculovirus yield and transgene expression in Sf9 cells. Biotechnol Lett 37(11):2159–2166

    Article  CAS  PubMed  Google Scholar 

  • Lu M, Iatrou K (1997) Characterization of a domain of the genome of BmNPV containing a functional gene for a small capsid protein and harboring deletions eliminating three open reading frames that are present in AcNPV. Gene 185(1):69–75

    Article  CAS  PubMed  Google Scholar 

  • McIntyre GJ, Fanning GC (2006) Design and cloning strategies for constructing shRNA expression vectors. Bmc Biotechnol 6(1):1–8

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mena JA, Kamen AA (2011) Insect cell technology is a versatile and robust vaccine manufacturing platform. Expert Rev Vaccines 10(7):1063–1081

    Article  CAS  PubMed  Google Scholar 

  • Nagai S, Alves CA, Kobayashi M, Ikeda M (2011) Comparative transient expression assay analysis of hycu-hr6- and IE1-dependent regulation of baculovirus gp64 early promoters in three insect cell lines. Virus Res 155(1):83–90

    Article  CAS  PubMed  Google Scholar 

  • Nagamine T, Abe A, Suzuki T, Dohmae N, Matsumoto S (2011) Co-expression of four baculovirus proteins, IE1, LEF3, P143, and PP31, elicits a cellular chromatin-containing reticulate structure in the nuclei of uninfected cells. Virology 417(1):188–195

    Article  CAS  PubMed  Google Scholar 

  • O’Reilly DR (1997) Use of baculovirus expression vectors. Methods Mol Biol 62:235–246

    PubMed  Google Scholar 

  • Oomens AG, Monsma SA, Blissard GW (1995) The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing. Virology 209(2):592–603

    Article  CAS  PubMed  Google Scholar 

  • Qi Q, Yao L, Liang Z, Yan D, Li Z, Huang Y, Sun J (2016) Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system. Mol Genet Genomics 291(6):2189–2198

    Article  CAS  PubMed  Google Scholar 

  • Rahman MM, Gopinathan KP (2003) Characterization of the gene encoding the envelope fusion glycoprotein GP64 from Bombyx mori nucleopolyhedrovirus. Virus Res 94(1):45–57

    Article  CAS  PubMed  Google Scholar 

  • Rankin C, Ladin BF, Weaver RF (1986) Physical mapping of temporally regulated, overlapping transcripts in the region of the 10K protein gene in Autographa californica nuclear polyhedrosis virus. J Virol 57(1):18–27

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rattanarojpong T, Khankaew S, Khunrae P, Vanichviriyakit R, Poomputsa K (2016) Recombinant baculovirus mediates dsRNA specific to rr2 delivery and its protective efficacy against WSSV infection. J Biotechnol 229:44–52

    Article  CAS  PubMed  Google Scholar 

  • Robalino J, Bartlett TC, Chapman RW, Gross PS, Browdy CL, Warr GW (2007) Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses. Dev Comp Immunol 31(6):539–547

    Article  CAS  PubMed  Google Scholar 

  • Saleh MC, Tassetto M, van Rij RP, Goic B, Gausson V, Berry B, Jacquier C, Antoniewski C, Andino R (2009) Antiviral immunity in Drosophila requires systemic RNA interference spread. Nature 458(7236):346–350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Senkevich TG, Koonin EV, Buller RM (1994) A poxvirus protein with a RING zinc finger motif is of crucial importance for virulence. Virology 198(1):118–128

    Article  CAS  PubMed  Google Scholar 

  • Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107(4):465–476

    Article  CAS  PubMed  Google Scholar 

  • Subbaiah EV, Royer C, Kanginakudru S, Satyavathi VV, Babu AS, Sivaprasad V, Chavancy G, Darocha M, Jalabert A, Mauchamp B, Basha I, Couble P, Nagaraju J (2013) Engineering silkworms for resistance to baculovirus through multigene RNA interference. Genetics 193(1):63–75

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun JC, Zhang EH, Yao LG, Zhang HL, Jin PF (2009) A high efficient method of constructing recombinant Bombyx mori (silkworm) multiple nucleopolyhedrovirus based on zero-background Tn7-mediated transposition in Escherichia coli. Biotechnol Prog 25(2):524–529

    Article  CAS  PubMed  Google Scholar 

  • Sun JC, Yao LG, Yao N, Xu H, Jin PF, Kan YC (2010) Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid. Biotechnol Appl Biochem 57(3):117–125

    Article  CAS  PubMed  Google Scholar 

  • Valdes VJ, Sampieri A, Sepulveda J, Vaca L (2003) Using double-stranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus. J Biol Chem 278(21):19317–19324

    Article  CAS  PubMed  Google Scholar 

  • Wang Q, Xie H, Zeng W, Wang L, Liu C, Wu J, Wang Y, Li Y, Bergmann SM (2018) Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells. Microb Pathog 114:68–74

    Article  CAS  PubMed  Google Scholar 

  • Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C, Pan G, Xu J, Liu C, Lin Y, Qian J, Hou Y, Wu Z, Li G, Pan M, Li C, Shen Y, Lan X, Yuan L, Li T, Xu H, Yang G, Wan Y, Zhu Y, Yu M, Shen W, Wu D, Xiang Z, Yu J, Wang J, Li R, Shi J, Li H, Li G, Su J, Wang X, Li G, Zhang Z, Wu Q, Li J, Zhang Q, Wei N, Xu J, Sun H, Dong L, Liu D, Zhao S, Zhao X, Meng Q, Lan F, Huang X, Li Y, Fang L, Li C, Li D, Sun Y, Zhang Z, Yang Z, Huang Y, Xi Y, Qi Q, He D, Huang H, Zhang X, Wang Z, Li W, Cao Y, Yu Y, Yu H, Li J, Ye J, Chen H, Zhou Y, Liu B, Wang J, Ye J, Ji H, Li S, Ni P, Zhang J, Zhang Y, Zheng H, Mao B, Wang W, Ye C, Li S, Wang J, Wong GK, Yang H (2004) A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science 306(5703):1937–1940

    Article  PubMed  Google Scholar 

  • Yao LG, Liu ZC, Zhang XM, Kan YC, Zhou JJ (2007) A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and large-scale expression of foreign proteins in silkworm (B. mori) larvae. Biotechnol Appl Biochem 48(Pt 1):45–53

    CAS  PubMed  Google Scholar 

  • Yao LG, Sun JC, Xu H, Kan YC, Zhang X, Yan HC (2010) A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli. J Biotechnol 145(1):23–29

    Article  CAS  PubMed  Google Scholar 

  • Yao LG, Jin PF, Su S, Xu H, He J, Peng L, Sun JC (2012a) Quantitative analysis of three commonly-used insect cell-specific promoters’ activities by transient and baculovirus-mediated expression. Afr J Biotechnol 11(5):1037–1045

    CAS  Google Scholar 

  • Yao LG, Wang S, Su S, Yao N, He J, Peng L, Sun JC (2012b) Construction of a baculovirus-silkworm multigene expression system and its application on producing virus-like particles. Plos One 7(3):e32510. https://doi.org/10.1371/journal.pone.0032510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101(1):25–33

    Article  CAS  PubMed  Google Scholar 

  • Zheng H, Wang X, Ren FF, Zou SL, Feng M, Xu LL, Yao LG, Sun JC (2018) Construction of a highly efficient display system for baculovirus and its application on multigene co-display. Mol Genet Genomics 293(5):1265–1277

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was funded by the National Natural Science Foundation of China (Grant Nos. 31872426, 31372373), the Natural Science Foundation of Guangdong Province, China (Grant No. 2016A030311018).

Author information

Author notes

  1. Hao Zheng and Feifei Ren contributed equally to this work.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding and Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, 510642, China

    Hao Zheng, Feifei Ren, Qiuyuan Lu, Zhenming Cao, Min Feng & Jingchen Sun

  2. Department of Animal Sciences, University of Manitoba, Winnipeg, MB, R3T2N2, Canada

    Jichen Song

  3. School of Life Sciences, Guangzhou University, Guangzhou, Guangdong, 510006, China

    Jisheng Liu

Authors
  1. Hao Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feifei Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiuyuan Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhenming Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jichen Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Min Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jisheng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jingchen Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

HZ and JS (J Sun) coordinated the project. HZ and FR performed the research. HZ JS (J Song) and JS (J Sun) wrote the manuscript. QL and JS (J Sun) contributed new methods and improved the manuscript. QL, ZC, and MF performed the data analysis. HZ, JL, and JS (J Sun) interpreted the context of results. All authors have read and approved the manuscript.

Corresponding authors

Correspondence to Min Feng or Jingchen Sun.

Ethics declarations

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

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

Additional information

Communicated by S. Hohmann.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zheng, H., Ren, F., Lu, Q. et al. An efficient method for multigene co-interference by recombinant Bombyx mori nucleopolyhedrovirus. Mol Genet Genomics 294, 111–120 (2019). https://doi.org/10.1007/s00438-018-1491-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-018-1491-9

Keywords

Search

Navigation