Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP‐antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP‐mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism.

Analysis ofthemechanism ofprotection intransgenic plants expressing thepotatovirus X coatprotein or its antisense RNA

Baculovirus expression vectors are frequently used to express glycoproteins, a subclass of proteins that includes many products with therapeutic value The insect cells that serve as hosts for baculovirus vector infection are capable of transferring oligosaccharide side chains (glycans) to the same sites in recombinant proteins as those that are used for native protein N-glycosylation in mammalian cells However, while mammalian cells produce compositionally more complex N-glycans containing terminal sialic acids, insect cells mostly produce simpler N-glycans with terminal mannose residues This structural difference between insect and mammalian N-glycans compromises the in vivo bioactivity of glycoproteins and can potentially induce allergenic reactions in humans These features obviously compromise the biomedical value of recombinant glycoproteins produced in the baculovirus expression vector system Thus, much effort has been expended to characterize the potential and limits of N-glycosylation in insect cell systems Discoveries from this research have led to the engineering of insect N-glycosylation pathways for assembly of mammalian-style glycans on baculovirus-expressed glycoproteins This chapter summarizes our knowledge of insect N-glycosylation pathways and describes efforts to engineer baculovirus vectors and insect cell lines to overcome the limits of insect cell glycosylation In addition, we consider other possible strategies for improving glycosylation in insect cells

Protein N‐Glycosylation in the Baculovirus–Insect Cell Expression System and Engineering of Insect Cells to Produce “Mammalianized” Recombinant Glycoproteins

The invention includes an expression vector engineered to produce double-stranded RNA (dsRNA) within a pest to be controlled. The dsRNA inhibits expression of at least one gene within the pest, wherein inhibition of the gene exerts a deleterious effect upon the pest. For example, inhibition of the gene can lead to cessation of feeding, growth, or development and can cause death of the pest. In a preferred embodiment of the invention the expression vector is a recombinant baculovirus that transcribes sense and antisense RNA under the control of the baculovirus IE-1 promoter and hr5 enhancer. Preferred genes to be inhibited include essential genes, genes involved in neurotransmission, and genes that are targets for conventional pesticides. The invention discloses baculovirus transfer plasmids useful for producing the recombinant baculovirus. The invention further discloses methods and formulations involving the expression vector.

Insect control agent

We have identified a gene required for strong expression of the polyhedrin gene by characterizing a mutant, tsB837, of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which is temperature sensitive (ts) for occluded virus production at the nonpermissive temperature. Marker rescue experiments utilizing an overlapping set of AcMNPV genomic clones revealed that the gene responsible for the ts mutant phenotype mapped to a region between 46 and 48 map units. Fragments (2.2 kb) from both wild-type AcMNPV and tsB837 genomes spanning the mutated region were sequenced, and a single nucleotide difference was observed. This mutation is predicted to substitute a single amino acid within a 44.4-kDa polypeptide. Analysis of protein synthesis in wild-type- and mutant-infected cells at the nonpermissive temperature indicated that polyhedrin synthesis was dramatically reduced in the mutant. Northern (RNA) blot analysis revealed that the mutant had markedly reduced levels of polyhedrin transcripts. Transcripts of another very late gene, p10, were also reduced but to a lesser degree. The transcription of two late genes (603 ORF and vp39) was neither reduced nor temporally delayed. Thus, the gene encoding this very late expression factor, designated vlf-1, regulates the levels of very late gene transcripts, and the tsB837 mutation affects the levels of polyhedrin gene transcripts more strongly than those of p10 transcripts. The product of the newly identified gene has a surprising but significant relationship to a family of integrases and resolvases.

Identification and characterization of vlf-1, a baculovirus gene involved in very late gene expression.

Baculoviruses are double-stranded DNA viruses which are highly selective for several insect groups. They are valuable natural control agents, but their utility in many agricultural applications has been limited by their slow speed of kill and narrow host specificity. Baculoviruses have been genetically modified to express foreign genes under powerful promoters in order to accelerate their speed of kill. In our and other laboratories, the expression of genes coding for insect juvenile hormone esterases and various peptide neurotoxins has resulted in recombinant baculoviruses with promise as biological insecticides. These viruses are efficacious in the laboratory, greenhouse and field and dramatically reduce damage caused by insect feeding. The recombinant viruses synergize and are synergized by classical pesticides such as pyrethroids. Since they are highly selective for pest insects, they can be used without disrupting biological control. Because the recombinant virus produces fewer progeny in infected larvae than the wild-type virus, they are rapidly out-competed in the ecosystem. The viruses can be used effectively with crops expressing endotoxins of Bacillus thuringiensis. They can be produced industrially but also by village industries, indicating that they have the potential to deliver sustainable pest control in developing countries. It remains to be seen, however, whether the current generation of recombinant baculoviruses will be competitive with the new generation of synthetic chemical pesticides. Current research clearly indicates, though, that the use of biological vectors of genes for insect control will find a place in agriculture. Baculoviruses will also prove valuable in testing the potential utility of proteins and peptides for insect control.

Recombinant baculoviruses for insect control.

The temporal expression of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes in Spodoptera frugiperda cells was studied using virus recombinants in which either gene was replaced by the juvenile hormone esterase (JHE) gene of Heliothis virescens. The JHE served as a highly specific and sensitive reporter for gene expression. Activation of the p10 gene followed a pattern different to that of polyhedrin. The p10 gene was activated a few hours earlier than the polyhedrin gene, but its expression reached a lower maximum level. Northern blot analysis complemented and confirmed the results obtained from the JHE assays. Co-infection of sense recombinants and those containing an anti-sense copy of the JHE gene in place of the polyhedrin or p10 gene resulted in reduced levels of JHE gene expression. These experiments independently supported the hypothesis that the p10 gene promoter is more active at earlier times post-infection than that of the polyhedrin gene. The results also highlight the potential of the antisense strategy as an experimental approach for the study of baculovirus gene regulation and possibly insect metabolism.

Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes.

Superior expression of intracellular and extracellular proteins from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 and polyhedrin promoters.

B. D. Hammock

Papers

Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes.

Superior expression of intracellular and extracellular proteins from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 and polyhedrin promoters.