Abstract
In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid. Bacmid DNA transfection assay revealed that the Bm101 knockout bacmid was unable to produce the infectious budded virus, while the Bm101 repair bacmid rescued this defect, allowing budded-virus titers to reach wild-type levels. Real time PCR analysis indicated that the viral DNA genome in the absence of Bm101 was unaffected in the first 24 h p.t. Thus, studies of a Bm101-null BACmid indicate that Bm101 is required for viral DNA replication during the infection cycle.
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References
Blissard GW, Wenz JR (1992) Baculovirus gp64 envelope glycoprotein is sufficient to mediate pH-dependent membrane fusion. J Virol 66:6829–6835
Chen H, Li M, Huang G, Mai W, Chen K, Zhou Y (2014) Bombyx mori nucleopolyhedrovirus ORF101 encodes a budded virus envelope associated protein. Curr Microbiol 69:158–163
Dai X, Stewart TM, Pathakamuri JA, Li Q, Theilmann DA (2004) Autographa californica multiple nucleopolyhedrovirus exon0 (orf141), which encodes a RING finger protein, is required for efficient production of budded virus. J Virol 78:9633–9644
de Jong J, Arif BM, Theilmann DA, Krell PJ (2009) Autographa californica multiple nucleopolyhedrovirus me53 (ac140) is a nonessential gene required for efficient budded-virus production. J Virol 83:7440–7448
Fang M, Dai X, Theilmann DA (2007) Autographa californica multiple nucleopolyhedrovirus EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus. J Virol 81:9859–9869
Granados RR, Lawler KA (1981) In vivo pathway of Autographa californica baculovirus invasion and infection. Virology 108:297–308
Hefferon KL, Oomens AG, Monsma SA, Finnerty CM, Blissard GW (1999) Host cell receptor binding by baculovirus GP64 and kinetics of virion entry. Virology 258:455–468
Herniou EA, Olszewski JA, Cory JS, O’Reilly DR (2003) The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48:211–234
Ke J, Wang J, Deng R, Wang X (2008) Autographa californica multiple nucleopolyhedrovirus ac66 is required for the efficient egress of nucleocapsids from the nucleus, general synthesis of preoccluded virions and occlusion body formation. Virology 374:421–431
Keddie BA, Aponte GW, Volkman LE (1989) The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Science 243:1728–1730
Luckow VA, Lee SC, Barry GF, Olins PO (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67:4566–4579
Lung OY, Cruz-Alvarez M, Blissard GW (2003) Ac23, an envelope fusion protein homolog in the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, is a viral pathogenicity factor. J Virol 77:328–339
McCarthy CB, Theilmann DA (2008) AcMNPV ac143 (odv-e18) is essential for mediating budded virus production and is the 30th baculovirus core gene. Virology 375:277–291
McCarthy CB, Dai X, Donly C, Theilmann DA (2008) Autographa californica multiple nucleopolyhedrovirus ac142, a core gene that is essential for BV production and ODV envelopment. Virology 372:325–339
Monsma SA, Blissard GW (1995) Identification of a membrane fusion domain and an oligomerization domain in the baculovirus GP64 envelope fusion protein. J Virol 69:2583–2595
Monsma SA, Oomens AG, Blissard GW (1996) The GP64 envelope fusion protein is an essential baculovirus protein required for cell-to-cell transmission of infection. J Virol 70:4607–4616
O’Reilly DR, Lk Miller, Luckow VE (1992) Baculovirus expression vectors: a laboratory manual. Freeman, NewYork
Olszewski J, Miller LK (1997) A role for baculovirus GP41 in budded virus production. Virology 233:292–301
Oomens AG, Blissard GW (1999) Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus. Virology 254:297–314
Pearson MN, Groten C, Rohrmann GF (2000) Identification of the lymantria dispar nucleopolyhedrovirus envelope fusion protein provides evidence for a phylogenetic division of the Baculoviridae. J Virol 74:6126–6131
Pijlman GP, Dortmans JC, Vermeesch AM, Yang K, Martens DE, Goldbach RW, Vlak JM (2002) Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses. J Virol 76:5605–5611
Thiem SM, Miller LK (1989) Identification, sequence, and transcriptional map** of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus. J Virol 63:2008–2018
Vanarsdall AL, Okano K, Rohrmann GF (2004) Characterization of a baculovirus with a deletion of vlf-1. Virology 326:191–201
Vanarsdall AL, Okano K, Rohrmann GF (2006) Characterization of the role of very late expression factor 1 in baculovirus capsid structure and DNA processing. J Virol 80:1724–1733
Wang M, Tan Y, Yin F, Deng F, Vlak JM, Hu Z, Wang H (2008) The F-like protein Ac23 enhances the infectivity of the budded virus of gp64-null Autographa californica multinucleocapsid nucleopolyhedrovirus pseudotyped with baculovirus envelope fusion protein F. J Virol 82:9800–9804
Wang M, Tuladhar E, Shen S, Wang H, van Oers MM, Vlak JM, Westenberg M (2010) Specificity of baculovirus P6.9 basic DNA-binding proteins and critical role of the C terminus in virion formation. J Virol 84:8821–8828
Westenberg M, Vlak JM (2008) GP64 of group I nucleopolyhedroviruses cannot readily rescue infectivity of group II f-null nucleopolyhedroviruses. J Gen Virol 89:424–431
Wu W, Lin T, Pan L, Yu M, Li Z, Pang Y, Yang K (2006) Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is interrupted upon deletion of the 38K gene. J Virol 80:11475–11485
Yamagishi J, Burnett ED, Harwood SH, Blissard GW (2007) The AcMNPV pp31 gene is not essential for productive AcMNPV replication or late gene transcription but appears to increase levels of most viral transcripts. Virology 365:34–47
Acknowledgments
This research was supported by the National Natural Science Foundation of China (31100118, 31370790), Natural Science Foundation of Jiangsu Province (BK2011495), Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (09KJB180001) and Startup Scientific Research Fund from Jiangsu University for Advanced Professionals (09JDG006).
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Chen, H., Li, M., Mai, W. et al. Analysis of BmNPV orf101 disruption: orf101 is essential for mediating budded virus production. Cytotechnology 66, 1021–1029 (2014). https://doi.org/10.1007/s10616-014-9772-6
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DOI: https://doi.org/10.1007/s10616-014-9772-6