Sequence analysis and expression of the polyhedrin gene of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV).

The segmented double-stranded RNA genome of Choristoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV) was extracted, polyadenylated, reverse-transcribed into cDNA and cloned. The cDNA clones that hybridized to the smallest genomic segment (segment 10) were identified, and its nucleotide sequence was determined. Genome segment 10 of CfCPV was found to be 1171 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 258 residues (Mr of 29,795), consistent with an apparent Mr of 30.5 kDa determined by SDS-PAGE of purified polyhedrin. Comparison of the nucleotide and amino acid sequences of the polyhedrin gene of CfCPV with those of other CPVs and with several nuclear polyhedrosis viruses revealed no particular homology. Analysis of the hydrophilic profiles and predicted secondary structures of Bombyx mori (BmCPV), Euxoa scandens (EsCPV) and CfCPV indicated the presence of seven similar regions located at the amino terminus of the polyhedrin polypeptide of the three viruses. The expression of the cloned CfCPV polyhedrin gene in Escherichia coli demonstrated that this polyhedrin has the property of self-assembly, since the production of crystal-like occlusion with a well-defined crystalline lattice structure was observed.

Human group II phospholipase A2 expressed in Trichoplusia ni larvae--isolation and kinetic properties of the enzyme.

Human secreted synovial fluid/platelet-type group II phospholipase A2 (sPLA2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 micrograms/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA2 showed a preference for substrate vesicles with a net negative charge. The baculovirus expression system provided active sPLA2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.

Horseradish peroxidase-labelled probes and enhanced chemiluminescence to detect baculoviruses in gypsy moth and eastern spruce budworm larvae.

Horseradish peroxidase-labelled whole genomic DNA probes and enhanced chemiluminescence procedures were utilized to detect baculovirus in insect macerates blotted on nylon membranes. Detection levels were similar to those found using 32P-labelled probes; 5 x 10(3) occlusion bodies (OBs), 2 x 10(3) OBs and 4 x 10(4) OBs of Lymantria dispar (L.) nuclear polyhedrosis virus (LdNPV), Choristoneura fumiferana (Clem.) NPV (CfNPV) and C. fumiferana granulosis virus (CfGV) respectively using 10 ng/ml LdNPV DNA probe and 20 ng/ml CfNPV and CfGV probe concentrations. Quantities of purified viral DNA detected were 0.56 ng LdNPV, 0.20 ng CfNPV and 0.10 ng CfGV at similar probe concentrations. Cross reactions were observed between LdNPV DNA probes and CfNPV. Multiple probing of membranes blotted with insect macerates was capable of diagnosing the presence of NPV and GV on membranes. This procedure appears to be useful in the diagnosis of large numbers of insects for several baculoviruses.