title: Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.
abstract: During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.|Graduate Program in Cell, Molecular and Structural Biology, Miami University, Oxford, OH 45056, USA.
authors.forename: Xin-Hua
authors.initials: XH
authors.lastname: Cheng
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Hillman
authors.affiliation: Agriculture Key Laboratory of Agricultural Entomology, Institute of Insect Science, Zhejiang University, Hangzhou 310058, PR China.
authors.forename: Chuan-Xi
authors.initials: CX
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Miami University, Oxford, OH 45056, USA.|Graduate Program in Cell, Molecular and Structural Biology, Miami University, Oxford, OH 45056, USA.
authors.forename: Xiao-Wen
authors.initials: XW
authors.lastname: Cheng
mesh_terms: D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism*; D000077183:Occlusion Body Matrix Proteins; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
chemical_list: C121638:FP25K protein, Autographa californica nucleopolyhedrovirus; D019590:Nucleocapsid Proteins; D000077183:Occlusion Body Matrix Proteins; D012333:RNA, Messenger; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D049452:Green Fluorescent Proteins; D000225:Adenine