H. Alan Wood

An agar overlay plaque assay method for autographa californica nuclear-polyhedrosis virus

A sensitive and reproducible infectivity assay of the Autographa californica nuclear-polyhedrosis virus (Ac-NPV) using Trichoplusia ni (TN-368) tissue culture cells was developed. A mixture of Ac-NPV infectious tissue culture supernatant and cells was centrifuged at 1000g for 60 min. The supernatant was removed, and the cells were overlaid with medium containing 1.5% agarose. Virus polyhedral inclusion bodies (IB) were observed in cells after 24 hr. Plaques (areas of IB containing cells) measured 2-3 mm after 5 days postinoculation. Plaque numbers were directly related to inoculum concentration. The assay was found to have a higher sensitivity than standard TCID 50 or methylcellulose overlay plaque assay procedures.

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.

Physical map and polyhedrin gene sequence of Lymantria dispar nuclear polyhedrosis virus

Restriction maps of the 166.6-kb genome of Lymantria dispar multiply-enveloped nuclear polyhedrosis virus (LdMNPV clone g) were constructed for BamHI, BglII, EcoRI, EcoRV, HindIII and KpnI, using cosmid pVK102 and pBluescript vectors. Southern hybridizations indicated that the LdMNPV genome contains five dispersed regions of intragenomic sequence homology. The polyhedrin gene of LdMNPV was located within BglII-E and the sequence of the 735-nucleotide (nt) coding region and 678 nt of flanking DNA was determined. A conserved 14-nt sequence, associated with transcriptional start points in other polyhedrins, was identified at 44 to 57 nt upstream from the start codon. The deduced polyhedrin amino acid (aa) sequence showed a high degree of homology with a previously determined protein sequence for LdMNPV polyhedrin (89%) and with deduced amino acid sequences for three other MNPV polyhedrins (74%). Optimal alignment of the four sequences indicated that LdMNPV polyhedrin possesses a single aa insertion at residue 4 and a single aa deletion at residue 164.

Inducing single-cell suspension of BTI-TN5B1-4 insect cells : I. The use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production

Sulfated polyanions have been successfully used to rapidly obtain and maintain stable single-cell suspension of BTI-TN5B1-4 cells, a cell line which has a high intrinsic capacity for recombinant protein production but clumps severely in suspension reducing its effectiveness as a host for foreign protein production with the baculovirus expression vector system. The efficacy of inducing single-cell suspension correlated positively with the increase in sulfation of the added polyanion. Unsulfated polyanions, neutral polymers, polycations, disaccharides, and monosaccharides were ineffective in inducing single-cell suspension.Elimination of clumping in suspension culture by adding a dispersing agent can lead to enhanced recombinant protein production. Inducing single-cell suspension with dextran sulfate, a highly sulfated polyanion, resulted in a four-fold increase in volumetric yield of the recombinant glycosylated protein, human secreted alkaline phosphatase, and a two-fold increase in volumetric yield of the recombinant cytoplasmic protein, beta-galactosidase. High yields of 82 U/ml (ca. 110 mg/L) for alkaline phosphatase, and 705 U/mL (ca. 2.3 g/L) for beta-galactosidase under elevated oxygen have been obtained. The optimum volumetric yield of alkaline phosphatase in BTI-TN5B1-4 dextran sulfate cells under elevated oxygen but unsupplemented medium is 6 to 11-fold higher than attached cultures, and 3-fold higher than the best yield obtained for SF21 cells in suspension at elevated oxygen and with nutrient supplementation. More importantly, cells can be infected at high density without complications from aggregation, which has important implications for scale-up.

Quantitative aspects of nuclear polyhedrosis virus infections in lepidopterous larvae: the dose-survival time relationship

Abstract Dose-survival time relationships are presented for neonate larvae of Heliothis zea inoculated with H. zea singly embedded nuclear polyhedrosis virus (HzSNPV) and Trichoplusia ni inoculated with the 1A clone of Autographa californica multiply embedded NPV, the HOB-mutant of this clone, or T. ni SNPV. For each of these virus-host combinations, plots of median survival time (ST) against logarithm of the inoculum concentration have the shape of an inverted sigmoid curve. The plateau of ST50 values at low doses is interpreted as resulting from the dose received by responding larvae remaining constant while the proportion of treated larvae receiving this dose decreases; evidence is presented that the plateau values at high doses are not produced by saturation of virus receptor sites on the midgut epithelium. A close similarity was noted between the dose-survival time relation of baculoviruses and their hosts on the one hand, and that of vertebrate pathogens and their hosts on the other; for the latter, mathematical modelswith predictive power have been developed.

Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: Influence of ammonia, time of harvest, temperature, and dissolved oxygen

Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of alpha-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetyl-hexosaminidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides containing terminal alpha(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.

Wound tumor virus: Purification and fractionation of the double-stranded ribonucleic acid

Abstract Double-phase phenol-SDS, single-phase phenol-SDS, SDS-urea, and sodium perchlorate methods were employed to isolate the ribonucleic acid (RNA) from purified wound tumor virus (WTV). The four different extraction procedures gave identical results. Seven components of WTV-RNA were identified by sucrose gradient analysis. However, fractionation of the RNA by polyacrylamide gel electrophoresis resulted in 8 distinct classes. Their average molecular weights were calculated to be 0.34, 0.58, 0.83, 1.02, 1.66, 1.98, 2.20, and 2.55 × 106. All components consisted of double-stranded RNA. Since the size and distribution of the WTV-RNA classes remained unchanged under all conditions tested, the 8 classes could not have arisen by random fragmentation.

BACULOVIRUS EXPRESSION OF AN INSECT GENE THAT ENCODES MULTIPLE NEUROPEPTIDES

Sex pheromone production in the corn earworm, Helicoverpa zea, is regulated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosynthesis activating neuropeptide). Hez-PBAN is encoded in a preprohormone that also contains four other structurally related peptides. Two recombinant baculoviruses that contain two different sequences of Hez-PBAN cDNA under the control of a strong polyhedrin promotor were constructed. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN coding sequence. The second virus, AcBX-PBAN, contains a synthetic chimera gene encoding a bombyxin signal peptide sequence fused to a pro-Hez-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected with AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromonotropic activity was not detected in the cells infected with AcWT-PBAN. Results of chromatographic and immunochemical studies showed that some of the potential processing sites in the expressed pro-Hez-PBAN sequence were not used during posttranslational processing in the AcBX-PBAN-4-infected BTI-TN-5B1-4 cells and 4th instar T. ni larvae. However, the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN-infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immunoreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-infected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significant reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient virus, Ac-E10.

Production of a Sialylated N-Linked Glycoprotein in Insect Cells

Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri‐ and tetra‐antennary complex N‐linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T‐flasks only performed incomplete oligosaccharide processing. The mechanism of HARV‐mediated changes in the eukaryotic N‐linked glycosylation pathway was investigated and could be mimicked under T‐flask growth conditions with the addition of N‐acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.

Monosaccharide compositions of Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) egg glycoproteins

Monosaccharide compositions of eggs from Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) were analyzed. Analyses were performed mainly with high performance anion exchange chromatography (HPAEC) using crude extracts of eggs or SDS-PAGE separated and PVDF-blotted protein bands. Man and GlcN were the major components in all cases, but low levels of Gal and Fuc were possibly present in some samples. Some T. ni egg glycoproteins even contained GalN. Although a peak comigrating with Neu5Ac could be detected with HPAEC-PAD or RP-HPLC (fluorometry) after derivatization with 1,2-diamino-4,5-methylenedioxy-benzene, the quantities were too small to be significant as an integral part of the analyzed glycoproteins. These data suggests that most of glycans on the glycoproteins are pauci-Man type N-glycans, but a small portion of N-glycan may be either hybrid type or complex type.