Article Figures & Data
Figures
- FIG 1
Temporal changes in P10 structures during AcMNPV infection of TN368 cells. (A) The amino acid sequence of AcMNPV P10 reveals three distinct regions, a coiled-coil domain at the N terminus (blue residues), a proline-rich region in the variable region (green residues), and a positively charged basic region at the C terminus (red residues) (R is arginine, and K is lysine). Amino acid residues of the heptad repeat in the coiled-coil region are denoted abcdefg, in which a and d are hydrophobic, whereas e and g are charged residues. (B) Wild-type virus-infected TN368 cells were analyzed at 48, 72, and 96 hpi by using confocal laser scanning microscopy. Cells were stained with anti-P10 and Alexa Fluor 488 antibodies to visualize P10 (green) and with anti-α-tubulin and Alexa Fluor 568 antibodies to visualize MTs (red). P10 and α-tubulin channels were merged to show coalignment. The position of the OB-filled nucleus is shown in the bright-field images. At 48 and 72 hpi, P10 filaments were coaligned with MTs and spanned the host cytoplasm; bundling of these filaments was evident at 72 hpi. P10 also formed perinuclear tubular structures that were present from 48 hpi and most developed at 96 hpi. The P10 cytoplasmic filaments appeared detached from the perinuclear tubule and partially disintegrated at 96 hpi. Bars, 30 μm.
- FIG 2
MALDI-TOF mass spectrometric analysis of the P10 C terminus. The AcMNPV P10 protein was harvested at 72 hpi and digested with endoproteinase GluC to cleave peptide bonds C terminally to glutamic acid residues. The peptide products were analyzed by MALDI-TOF MS (Ultraflex; Bruker Daltonics) in the linear mode. The image shows a portion of the spectrum containing the peptides of interest from the P10 C terminus. The x axis represents m/z values, and the y axis represents the absolute intensity. Peaks with m/z values of 1,475.81 and 1,555.75 corresponded to the non- and monophosphorylated states of the P10 C-terminal peptide 82LDSDARRGKRSSK94. a.u., arbitrary units.
- FIG 3
Construction of recombinant viruses. (A) Wild-type or mutant p10 flanked by XbaI and XmaI restriction sites was inserted downstream of the polyhedrin promoter in the transfer vector pBacPAK8. Recombinant baculoviruses were made by allowing homologous recombination of the transfer vector and flashBACULTRA. Four viruses were constructed; in the single mutants AcP10S92A and AcP10S93A, serines 92 and 93 were mutated to alanine, respectively. In the double mutant AcP10S9293A, both serines 92 and 93 were mutated to alanine. AcP10wt contained wild-type p10. (B) pAcUW2B was used to construct the His-tagged wild-type and mutant p10-carrying viruses. This vector included a complete polh gene. The P10 fragment was inserted downstream of the P10 promoter in pAcUW2B by using the PstI and SpeI restriction sites. Six histidine residues followed by the TEV cleavage site residues were added at the N terminus. Two recombinant viruses were constructed by cotransfecting pAcUW2B-modified vectors with flashBACULTRA: AcUW2B-His-P10wt, containing the wild-type p10 gene, and AcUW2B-His-P10S93A. The displayed genes are not to scale.
- FIG 4
Analysis of wild-type and mutant P10 structures. TN368 cells were infected with AcP10wt, AcP10S92A, AcP10S93A, or AcP10S9293A and then fixed at 72 and 96 hpi. P10 structures were visualized by using anti-P10- and Alexa Fluor 488 antibodies; microtubules (red) were visualized by using anti-α-tubulin and Alexa Fluor 568 antibodies. P10 and α-tubulin channels were merged to show coalignment. At 72 hpi, cells infected with AcP10wt or AcP10S92A showed both P10 perinuclear tubules (NT) and cytoplasmic filaments (CF). By 96 hpi, the perinuclear tubules had matured, and most cytoplasmic filaments were detached from the central tubule. Cells infected with AcP10S93A or AcP10S9293A lacked perinuclear tubules and displayed rigid and angular cytoplasmic filaments that were not fully detached from the nucleus. Images are representative. Bars, 30 μm.
- FIG 5
MALDI-TOF mass spectrometric analysis of the P10 peptides from AcP10wt and AcP10S93A. In-gel digestion of P10 proteins (separated by SDS-PAGE) from AcP10wt and AcP10S93A was carried out with endoproteinase GluC; this cleaved peptide bonds C terminally to glutamic acid residues in ammonium carbonate buffer. The peptide fragments were analyzed by MALDI-TOF MS (Ultraflex; Bruker Daltonics) in the linear mode. The image shows a portion of the spectrum containing the P10 C-terminal peptides of interest. The x axis represents m/z values, and the y axis represents absolute intensity as measured by the detector. The top panel shows the MALDI-TOF spectrum of the P10 C-terminal peptide from AcP10wt, in which wild-type P10 expression was driven by the polyhedrin gene promoter. The MALDI-TOF spectrum shows peaks with m/z values of 1,475.81 and 1,555.75 that corresponded to the non- and monophosphorylated states of the P10 peptide 82LDSDARRGKRSSK94. The bottom panel shows the MALDI-TOF spectrum of the P10 peptide from AcP10S93A. In this recombinant virus, the P10 serine 93 residue was mutated to alanine, and mutant expression was driven by the polh promoter. The MALDI-TOF spectrum shows a signal at [M + H]+ 1,459.85, corresponding to the peptide 82LDSDARRGKRSAK94; however, no phosphorylated form of this peptide was observed (no signal at m/z 1,539).
- FIG 6
Secondary structure of wild-type P10 and its serine 93 mutant. Spectra were averaged from 4 to 16 scans in the wavelength range of 260 to 190 nm. CD was measured in ellipticity units of millidegrees (mdeg). The CD spectra of the serine 93 mutant and wild-type P10 revealed differences in the minima. The table shows the percentages of different secondary structures in the two proteins following LINCOMB analysis of spectra.
Tables
- TABLE 1
Recently identified P10 homologues
Baculovirus GenBank accession no. Alphabaculoviruses Adoxophyes orana nucleopolyhedrovirus YP_002300549 Agrotis ipsilon multiple nucleopolyhedrovirus YP_002268180 Ampelophaga rubiginosa nucleopolyhedrovirus ACB32175 Apocheima cinerarium nucleopolyhedrovirus YP_006607870 Bombyx mandarina nucleopolyhedrovirus ACQ57311 Choristoneura murinana alphabaculovirus AHD25508 Choristoneura occidentalis alphabaculovirus AGR56909 Choristoneura rosaceana alphabaculovirus YP_008378374 Euproctis pseudoconspersa nucleopolyhedrovirus ACO53500 Hemileuca sp. nucleopolyhedrovirus YP_008378239 Lambdina fiscellaria nucleopolyhedrovirus YP_009133322 Lonomia obliqua multiple nucleopolyhedrovirus AKN80950 Mamestra brassicae multiple nucleopolyhedrovirus YP_009011213 Orgyia leucostigma nucleopolyhedrovirus ABY65747 Philosamia cynthia ricini nucleopolyhedrovirus virus AFY62828 Pseudoplusia includens single nucleopolyhedrovirus IE AJD80709 Spodoptera littoralis nucleopolyhedrovirus AGE89872 Sucra jujuba nucleopolyhedrovirus YP_009186700 Thysanoplusia orichalcea nucleopolyhedrovirus YP_007250541 Betabaculoviruses Clostera anachoreta granulovirus AEB00304 Clostera anastomosis granulovirus AKS25391 Erinnyis ello granulovirus YP_009091893 Mocis sp. granulovirus AKR17400 Pieris rapae granulovirus ACZ63503 Pseudaletia unipuncta granulovirus YP_003422344 - TABLE 2
PCR primers
Primer Sequence P10_S92AF 5′-CGTGGTAAACGCGCGTCCAAGTAAGAATTC-3′ P10_S92AR 5′-TTCTTACTTGGACGCGCGTTTACCACGAC-3′ P10_S93AF 5′-GGTAAACGCGCGGCGAAGTAAGAATTCG-3′ P10_S93AR 5′-GAATTCTTACTTCGCCGCGCGTTTACCAC-3′ P10_S9293A_pBP8F 5′-CATGTAGCTCTAGAATGTCAAAGCCTAACG-3′ P10_S9293A_pBP8R 5′-CATGTAGCCCCGGGTTACTTCGCCGCGC-3′ P10_S92A_pBP8F 5′-CGTCGTGGTAAACGCGCTTCCAAGTAACCCGGG-3′ P10_S92A_pBP8R 5′-CCCGGGTTACTTGGAAGCGCGTTTACCACGACG-3′ P10_S93A_pBP8F 5′-CTCGTCGTGGTAAACGCAGTGCCAAGTAACCC-3′ P10_S93A_pBP8R 5′-GGGTTACTTGGCACTGCGTTTACCACGACGAG-3′ P10_wtF 5′-CATGTAGCTCTAGAATGTCAAAGCCTAACG-3′ P10_wtR 5′-CATGTAGCCCCGGGTTACTTGGAACTGCG-3′ P10_wt_pW2BF 5′-GTAGCCTGCAGTTACTTGGAACTGCGTTTACC-3′ P10_wt_pW2BR 5′-GTAGCAAGCCTATGTCAAAGCCTAACGTTTTGACG-3′ P10_S93A_pW2BF 5′-GCTCTGCAGTTACTTGGCACTGCGTTTACCACGAC-3′ P10_S93A_pW2BR 5′-GTCGTGGTAAACGCAGTGCCAAGTAACTGCAGAGC-3′ HISP10_wt_pW2BF 5′-GTAGCCTGCAGTTACTTGGAACTGCGTTTACCACGACGAGCGTC-3′ HISP10_wt_pW2BR 5′-GCACTAGTATGCATCACCATCACCATCACGAAAACCTGTATTTTCAGGGCTCAAAGCCTAACG-3′ HISP10_S93A_pW2BF 5′-GTAGCCTGCAGTTACTTGGAACTGCGTTTACCACGACGAGCGTC-3′ HISP10_S93A_pW2BR 5′-GTAGCCTGCAGTTACTTGGCACTGCGTTTACCACGACGAGC-3′
