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.

Baculovirus FP25K Localization: Role of the Coiled-Coil Domain

“…Screening and selection of AcBacmid clones with an inactivated endogenous fp25k (AcBac-fp::287) were performed by colony PCR with the above-described fp25k PCR primers and conditions. The fp25k locus of AcBac-fp::287 was mutated through the insertion of a 287-bp piece of Sf21 host DNA into the fp25k coding sequence at the TTAA site at nucleotide (nt) ϩ425, as previously reported (10). With a premature stop codon, this FP25K mutant sequence contains a 73-amino-acid (aa) deletion at the C terminus.…”

“…Viral DNA was extracted from passaged AcBacmid BV. PCR was performed on passaged AcBacmid BV DNA by using fp25k gene-specific primers fp25k-F and fp25k-R (Table 1) with expected 1.2-kb (wild-type) and 1.5-kb (mutant) fp25k product sizes (10). The BV of the passage that showed a prominent 1.5-kb PCR product (passage 10) was used for viral DNA extraction and eventual transformation of E. coli DH10B cells.…”

“…BV was harvested by collection of culture medium, followed by centrifugation at 500 ϫ g for 5 min to remove cell debris. To generate a virus that produces nonfused GFP, the gfp coding sequence (CDS) was cloned from pBlueGFP into the pFastBac1 vector, and the resulting construct, pFastBac1-GFP, was transformed into competent DH10Bac bacteria to produce AcBacGFP in insect cells (10). Titers of all recombinant viruses, including viruses used for the setup of the time course study described below, were determined by endpoint dilution assays (50% tissue culture infective dose [TCID 50 ]) as described previously (34).…”

“…In contrast, nucleocapsids destined to remain in the nucleus acquire an envelope from blebbing of the inner nuclear membrane (INM) to form ODVs (8,9). This membraneblebbing process is partially facilitated by a particular 25-kDa nu-cleocapsid protein, FP25K (8,10,11). During this late phase of infection, occlusion bodies (OBs) are formed around ODVs with either the matrix protein polyhedrin or granulin, in the case of alpha-and gammabaculoviruses or betabaculoviruses, respectively (3,4,8,9).…”

“…The process of INM blebbing, facilitated by FP25K and other ODV envelope proteins, is thought to be a general switch from BV to ODV production (14)(15)(16). This multifunctional FP25K protein is not required for infection in vitro, but it is necessary in vivo for proper ODV envelopment, virion occlusion, and insect per os infectivity (10,(16)(17)(18)(19).…”