Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene

Abstract

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

FIG. 1.

Generation of recombinant BmNPVs expressing fp25K genes from BmNPV, AcMNPV, SpltMNPV, and XecnGV. (A) Alignment of FP25K proteins from BmNPV, AcMNPV, SpltMNPV, and XecnGV (XcGV). In the sequences shown, black shading denotes identical residues and gray shading indicates similarities among the four FP25K proteins. (B) Schematic representation of recombinant BmNPVs. The set of PCR primers (FP_CF1 and FP_CR1) used in genotyping experiments is also shown. ORF, open reading frame. (C) PCR analysis of the genomes of T3, Bm25KD (25KD), Bm25KD-Bm (25KD-Bm), Bm25KD-Ac (25KD-Ac), Bm25KD-Splt (25KD-Splt), and Bm25KD-Xc (25KD-Xc). Each genotype was confirmed by PCR using the primers FP_CF1and FP_CR1. (D) Expression of His-tagged FP25K proteins in BmNPV-infected BmN cells. BmN cells were infected with T3 or recombinant BmNPVs, harvested at 3 dpi, and subjected to Western blot analysis with anti-His antibody. The gel stained with Coomassie brilliant blue (CBB) is shown in the lower panel. The molecular masses of protein standards are indicated to the right. The arrowhead indicates polyhedrin.

FIG. 2.

Comparison of OB production levels in BmN cells. (A) Light microscopic observations of BmNPV-infected BmN cells at 5 dpi. (B) OB production. BmNPV-infected BmN cells at 3 dpi were gently scraped using a rubber policeman, and total OB production was measured. Data shown are means ± standard deviations (SD; n = 3). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control. (C) polh expression in Bm25KD-Bm- and Bm25KD-Ac-infected BmN cells. BmN cells were infected with Bm25KD-Bm and Bm25KD-Ac at an MOI of 5. Total RNA was reverse transcribed, and qRT-PCR analysis of polh was performed. Data shown are means ± SD (n = 3). *, P < 0.05 by Student's t test. (D) OB release from BmNPV-infected BmN cells at 5 dpi. Data shown are means ± SD (n = 3). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control.

FIG. 3.

Comparison of BV production levels and plaque sizes in BmN cells. (A) BV production. BmN cells were infected with T3 or recombinant BmNPVs at an MOI of 5. BV titers at 1 and 2 dpi were determined by plaque assays. Data shown are means ± SD (n = 3). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control. (B) Plaque size. Plaques were photographed, and their sizes were quantified using ImageJ software. Data shown are means ± SD (n = 10). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control.

FIG. 4.

Effects of substitution of fp25K genes on in vivo infection phenotypes. (A) Postmortem host degradation of BmNPV-infected larvae. B. mori larvae were subcutaneously injected with BVs from T3 or recombinant BmNPVs. Host degradation was assessed visually, and infected larvae were photographed at 7 dpi. (B) V-CATH secretion into the hemolymph of BmNPV-infected larvae. B. mori larvae were injected with BVs from T3 or recombinant BmNPVs, and hemolymph samples were collected at 4 dpi. Western blot analysis of the hemolymph samples was performed with anti-V-CATH antibody. The molecular masses of protein standards are indicated to the left. P, pro-V-CATH; M, mature form of V-CATH. (C) V-CATH activities of hemolymph samples from virus-infected larvae. Hemolymph samples from T3- or recombinant BmNPV-infected larvae at 4 dpi were assayed for V-CATH activity in the presence (black bars) or absence (white bars) of E64, a cysteine protease inhibitor. Data shown are means ± SD (n = 10). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control. (D) Quantification of OB release into the hemolymph of BmNPV-infected larvae at 4 dpi. Data shown are means ± SD (n = 10). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using T3 as a control.

FIG. 5.

Generation of recombinant BmNPVs expressing chimeric fp25K genes. (A) Diagrammatic representation of chimeric FP25Ks. Four viruses (BmAc1, BmAc2, AcBm1, and AcBm2) expressing chimeras generated from BmNPV and AcMNPV FP25Ks were constructed. Arrowheads indicate the amino acid residues in BmNPV and AcMNPV FP25Ks that are not identical. (B) Expression of His-tagged chimeric FP25K proteins in BmNPV-infected BmN cells. BmN cells infected with recombinant BmNPVs were collected and subjected to Western blot analysis with anti-His antibody. The molecular masses of protein standards are indicated to the left. (C) OB production. BmNPV-infected BmN cells at 3 dpi were gently scraped using a rubber policeman, and total OB production was measured. Data shown are means ± SD (n = 3). *, P < 0.05 by one-way ANOVA and Dunnett's posttests using Bm25KD-Bm as a control.

FIG. 6.

Expression of mIL-3 and luciferase using a novel BmNPV vector with AcMNPV fp25K. (A) Expression of mIL-3 in BmN cells. BmN cells were infected with IL-Bm and IL-Ac and harvested at 1 to 3 dpi in cell lysis buffer. The lysates were then analyzed by Western blotting with anti-FLAG antibody. Western blotting using anti-actin was also performed. The molecular masses of protein standards are indicated to the left. (B) Secretion of mIL-3 in BmN cells. BmN cells were infected with IL-Bm and IL-Ac, and the culture medium was harvested at 1 to 3 dpi. Western blotting of the medium samples was performed with anti-FLAG antibody. The molecular masses of protein standards are indicated to the left. (C) Secretion of mIL-3 into the larval hemolymph. B. mori larvae were injected with BVs from IL-Bm and IL-Ac, and hemolymph samples were collected at 2 to 4 dpi. Western blot analysis of the hemolymph samples was performed with anti-FLAG antibody. (D) Expression of luciferase in BmN cells. BmN cells were infected with Luc-Bm and Luc-Ac and harvested at 1 to 3 dpi in cell lysis buffer. The lysates were then subjected to a luciferase assay. Data shown are means ± SD (n = 3). *, P < 0.05 by Student's t test.