Lois K. Miller

Bacterial, Viral, and Fungal Insecticides

Microorganisms that are pathogenic to insects provide a wealth of biological material that can be exploited by humans to control insect pests. Innovative applications of a few such entomopathogens are found throughout the world, but widespread commercial production of microbial insecticides awaits further studies of the biology, ecology, and pathogenicity of the agents. Genetic engineering techniques may be used to increase the virulence of these microorganisms, as well as to make them more tolerant of physical and chemical conditions and perhaps to broaden their host ranges. The use of microbial insecticides could decrease our dependence on chemical pesticides.

Changes in the nucleoprotein complexes of a baculovirus DNA during infection

The nature of the DNA-protein complexes assumed by Autographa californica nuclear polyhedrosis virus (AcNPV) DNA during infection of Spodoptera frugiperda cells was investigated by micrococcal nuclease digestion of infected nuclei. Both parental viral DNA and progeny viral DNA assumed a chromatin-like structure early in infection. By late times (24 hr) p.i., the viral DNA acquired a unique nucleoprotein structure. In addition to fragments of mononucleosome size (185 bp), two subnucleosomal bands of 120 and 90 bp were observed. The subnucleosomal bands contained exclusively viral DNA. No alteration in the nature of the host chromatin structure following AcNPV infection was observed. An examination of the basic chromatin-associated proteins revealed two major viral-induced proteins having molecular weights of 15K and 39K. The induction of the basic 15K protein between 10 and 24 hr p.i. coincided with the appearance of the altered nucleoprotein structure observed by 24 hr p.i. and the cessation of histone synthesis.

A temperature-sensitive mutant of the baculovirus Autographa californica nuclear polyhedrosis virus defective in an early function required for further gene expression.

Fifteen temperature-sensitive (ts) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) have been analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins synthesized in infected cells. One of the mutants, tsB821, was found to be defective in a very early function. Seven virus-induced proteins were synthesized by 2 hr postinfection. In marked contrast to wild-type virus and the other 14 ts mutants, the synthesis of further virus-induced proteins did not occur in tsB821-infected cells at the restrictive temperature (33 degrees ). Host protein synthesis continued as normal after transient expression of the seven early proteins. Viral-specific DNA synthesis was blocked or significantly delayed in tsB821-infected cells at 33 degrees . The relative synthesis of certain viral-induced proteins, particularly P31, P32, P42, P66, and P69, varied considerably in the remaining 14 mutants at 33 degrees. Three mutants exhibited alterations in specific polypeptides; P75 was approximately 1 kDa smaller in tsB1075, P40 was approximately 1 kDa smaller in tsB951, and P25 was greatly reduced in quantity or altered in tsB305.

Variation m the temporal expression of overlapping baculovirus transcripts

To investigate gene expression from the Autographa californica nuclear polyhydrolysis virus genome (AcNPV), complementary DNA (cDNA) was synthesized from polyadenylated RNA transcribed at 2 h and 10 h postinfection (p.i.) and then cloned into Escherichia coli using plasmid pUC-9. Eighteen 2 h cDNA plasmids were homologous to five distinct regions of the viral genome, while forty-nine 10 h cDNA plasmids were homologous to 15 regions including the five regions transcribed at 2 h. Temporal expression of polyadenylated RNA transcribed from diverse regions of the genome was examined using Northern blot hybridization with the above 2 and 10 h cDNA probes. All regions displayed overlapping sets of RNAs. In addition to HindIII-I/EcoRI-F (IF) and HindIII-B2/EcoRI-H (B2H), several, but not all, regions showed a sequential appearance of higher molecular weight RNAs as the infection progressed. Each overlapping set of RNAs exhibited unique characteristics including variations in the number of overlapping transcripts, their temporal regulation, and their relative abundance during the course of infection.

CORRELATING GENETIC MUTATIONS OF A BACULOVIRUS WITH THE PHYSICAL MAP OF THE DNA GENOME

The ability to map temperature-sensitive mutations of a baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV), has been demonstrated. A marker rescue technique, based on the ability of a restriction endonuclease fragment of wild-type AcNPV L-1 DNA to re-combine with a ts mutant DNA genome was employed to determine approximate locations of several ts mutations of AcNPV with respect to the physical restriction endonuclease fragment map of AcNPV L-l DNA. Two mutants, ts N332 and ts B837, are defective in occlusion but non-defective in extracellular non-occluded virus formation. Thus, scoring for rescue of these mutants by wild-type fragments was based on visualizing occlusion bodies within plaques produced at the restrictive temperature following transfection. In addition, five plaque morphology mutants producing few polyhedra per cell, FPs, were isolated after serial passage of AcNPV in TN-368 cells. Analysis of the DNA genomes of these mutants with restriction endonucleases revealed that one mutant, FP-D, contains an additional sequence of DNA located at 66 md units (75%) on the AcNPV L-1 physical map.

A new variant of Autographa californica nuclear polyhedrosis virus

Abstract A variant of the baculovirus, Autographa californica nuclear polyhedrosis virus, has been isolated in Idaho during an epizootic disease in a field population of A. californica. Genotypic characterization indicates that the virus is distinct from variants previously characterized. Analysis of five clones, derived by plaque purification in cell culture, indicates relative homogeneity of the original virus isolate. Further exploration of the factors involved in natural genetic variability of baculoviruses is appropriate.

An Insect Virus for Genetic Engineering: Developing Baculovirus Polyhedrin Substitution Vectors

Baculoviruses are exceptionally attractive candidates as vectors for propagating and expressing exogenous DNAs in a eukaryotic (invertebrate) environment (Miller, 1981a). Among the features which make baculoviruses highly advantageous as recombinant DNA vector systems are (1) a covalently-closed, circular, nuclear-replicating DNA genome, (2) an extendable rod-shaped capsid, (3) a group of genes, involved in occlusion, that are nonessential for infectious virus production and thus deletable, and (4) a strong promoter which is turned on after infectious virus production and controls the synthesis of the major occlusion body protein (poly-hedrin), constituting approximately ten percent of the protein of infected cells. The replacement of the polyhedrin gene with passenger DNA was previously suggested as an approach to using baculoviruses as recombinant DNA vectors (Miller, 1981a). The initial experimental advances our laboratory has made in developing the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector in insect cells are described herein.