title: Removal of transposon target sites from the Autographa californica multiple nucleopolyhedrovirus fp25k gene delays, but does not prevent, accumulation of the few polyhedra phenotype.
abstract: Low-cost, large-scale production of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) using continuous insect cell culture is seriously hindered by the accumulation of AcMNPV mutants. Specifically, few-polyhedra (FP) mutants, with a reduced yield of occluded virus (polyhedra) and decreased infectivity, usually accumulate upon passaging in cell culture. FP mutations result from transposon insertions in the baculovirus fp25k gene, leading to significantly reduced levels of FP25K protein synthesis. This study evaluated the effects of removing the transposon insertion sites from the wild-type baculovirus fp25k gene; the mutated virus was denoted Ac-FPm. Specifically, this study involved a detailed comparison of wild-type (WT) AcMNPV and Ac-FPm with regard to the proportion of cells having polyhedra, number of polyhedra per cell, the fraction of empty polyhedra, number of occlusion-derived viruses per polyhedron, number of nucleocapsids in the nuclei, FP25K protein synthesis and genetic analysis of the fp25k gene. Removal of TTAA transposon insertion sites from the fp25k gene stabilized FP25K protein synthesis and delayed the appearance of the FP phenotype from passage 5 to passage 10. Electron micrographs revealed that more virus particles were found inside the nuclei of cells infected with Ac-FPm than in the nuclei of cells infected with WT AcMNPV (at passage 10). Abnormalities, however, were observed in envelopment of nucleocapsids and virus particle occlusion within Ac-FPm polyhedra. Thus, the FP phenotype appeared in spite of continued FP25K protein synthesis, suggesting that mechanisms other than fp25k gene disruption can lead to the FP phenotype.
authors.affiliation: University of Iowa, Department of Chemical and Biochemical Engineering, Iowa City, IA 52242-1527, USA.
authors.forename: Lopamudra
authors.initials: L
authors.lastname: Giri
authors.forename: Huarang
authors.initials: H
authors.lastname: Li
authors.forename: David
authors.initials: D
authors.lastname: Sandgren
authors.forename: Michael G
authors.initials: MG
authors.lastname: Feiss
authors.forename: Richard
authors.initials: R
authors.lastname: Roller
authors.forename: Bryony C
authors.initials: BC
authors.lastname: Bonning
authors.forename: David W
authors.initials: DW
authors.lastname: Murhammer
mesh_terms: D000818:Animals; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics*; D007915:Lepidoptera / Q000821:virology*; D016254:Mutagenesis, Insertional; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D010641:Phenotype; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
chemical_list: D004251:DNA Transposable Elements; D004279:DNA, Viral; D014764:Viral Proteins
references.citation: Virology. 1985 Sep;145(2):356-61
references.pmid: 2992159
references.citation: J Virol. 1983 Aug;47(2):287-300
references.pmid: 16789244
references.citation: J Gen Virol. 2005 Sep;86(Pt 9):2439-2444
references.pmid: 16099901
references.citation: J Gen Virol. 2006 May;87(Pt 5):1259-1265
references.pmid: 16603528
references.citation: J Virol. 1997 Feb;71(2):1097-106
references.pmid: 8995630
references.citation: Virology. 1996 Dec 1;226(1):34-46
references.pmid: 8941320
references.citation: J Virol. 2010 May;84(10):5191-200
references.pmid: 20219904
references.citation: Biotechnol Prog. 2003 Jan-Feb;19(1):238-42
references.pmid: 12573032
references.citation: Virus Res. 1987 Jun;7(4):335-49
references.pmid: 2887077
references.citation: J Gen Virol. 1990 May;71 ( Pt 5):1029-37
references.pmid: 2189022
references.citation: Virology. 1991 Aug;183(2):739-46
references.pmid: 1853572
references.citation: Virology. 1995 Apr 1;208(1):279-88
references.pmid: 11831709
references.citation: J Virol. 1975 Jan;17(1):254-68
references.pmid: 1107597
references.citation: Virology. 1989 Sep;172(1):156-69
references.pmid: 2549707
references.citation: Virology. 1987 Nov;161(1):8-17
references.pmid: 2823472
references.citation: Arch Virol. 2009;154(3):445-56
references.pmid: 19221693
references.citation: Virus Res. 2008 May;133(2):157-66
references.pmid: 18261819
references.citation: Virology. 1998 Jan 5;240(1):64-75
references.pmid: 9448690
references.citation: J Ultrastruct Res. 1974 Dec;49(3):419-35
references.pmid: 4616102
references.citation: J Gen Virol. 2002 Apr;83(Pt 4):945-955
references.pmid: 11907345
references.citation: Arch Virol. 2003 Mar;148(3):587-97
references.pmid: 12607109
references.citation: Virology. 1990 Feb;174(2):354-63
references.pmid: 2154879
references.citation: J Virol. 2001 Nov;75(22):10829-42
references.pmid: 11602724
references.citation: Appl Microbiol Biotechnol. 1990 Apr;33(1):43-7
references.pmid: 1366563
references.citation: Arch Virol. 2000;145(4):827-34
references.pmid: 10893160
references.citation: J Virol. 1995 Jan;69(1):156-65
references.pmid: 7983706
references.citation: Arch Virol. 1999;144(7):1275-85
references.pmid: 10481736
references.citation: J Gen Virol. 1995 Jun;76 ( Pt 6):1451-9
references.pmid: 7782773
references.citation: J Gen Virol. 1996 Jan;77 ( Pt 1):147-53
references.pmid: 8558123
references.citation: Virology. 1996 Feb 1;216(1):235-7
references.pmid: 8614994
references.citation: Mol Cell Biol. 1990 Jun;10(6):3067-77
references.pmid: 1692964
references.citation: Virology. 1988 Jan;162(1):206-20
references.pmid: 2829419
references.citation: Cytotechnology. 1996 Jan;20(1-3):291-7
references.pmid: 22358492
references.citation: J Virol. 1999 Oct;73(10):8559-70
references.pmid: 10482609
references.citation: Virology. 1982 Mar;117(2):366-78
references.pmid: 18635122
references.citation: Virology. 2006 Apr 10;347(2):261-76
references.pmid: 16427676
references.citation: In Vitro. 1977 Apr;13(4):213-7
references.pmid: 68913
references.citation: J Invertebr Pathol. 2000 Jul;76(1):13-9
references.pmid: 10963398
references.citation: J Virol. 1976 Jun;18(3):1040-50
references.pmid: 775129
references.citation: J Virol. 1992 Dec;66(12):6903-11
references.pmid: 1433499
references.citation: Gene. 1989 Sep 1;81(1):97-108
references.pmid: 2553540
references.citation: Nature. 1982 Oct 7;299(5883):562-4
references.pmid: 6289125
references.citation: Virology. 1994 Aug 1;202(2):586-605
references.pmid: 8030224
references.citation: J Invertebr Pathol. 2009 Mar;100(3):153-9
references.pmid: 19135449
references.citation: Annu Rev Entomol. 1999;44:257-89
references.pmid: 15012374