Summary
The requirements for high level expression of three foreign proteins using the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV, Baculoviridae) have been investigated. In Spodoptera frugiperda cells infected with the appropriate recombinant baculoviruses, the synthesis of the two S RNA coded genes of lymphocytic choriomeningitis virus (LCMV; i.e. the nucleoprotein, N, and glycoprotein precursor, GPC), or the haemagglutinin gene of influenza A virus, appears to be related to the degree of integrity of the 5′ upstream sequence of the polyhedrin gene. No effect on the level of N protein expression was detected when all the polyhedrin gene coding sequences or some of the immediate 3′ downstream sequences were deleted. Using the most efficient expression viruses derived from a new transfer vector, pAcYM1, it has been estimated that LCMV N protein represented approximately 50% of the total cellular protein, an observation consistent with the presence of numerous inclusion bodies in the cytoplasm of infected cells. For recombinant viruses derived from the pAcYM1 transfer vector containing the LCMV GPC gene, the level of synthesis of the arenavirus glycoprotein was equivalent to approximately 20% of the cellular protein. Thin sections of cells infected with the GPC recombinant revealed a highly vacuolated cytoplasm.

Baculovirus Expression Vectors: the Requirements for High Level Expression of Proteins, Including Glycoproteins

A soluble receptor for interleukin-1β encoded by vaccinia virus: A novel mechanism of virus modulation of the host response to infection

We describe a method to introduce site-specific mutations into the genome of Autographa californica nuclear polyhedrosis virus. Specifically, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the major protein that forms viral occlusions in infected cells, was mutagenized by introducing deletions into the cloned DNA fragment containing the gene. The mutagenized polyhedrin gene was transferred to the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants that had undergone allelic exchange. Recombinant viruses with mutant polyhedrin genes were obtained by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants demonstrated that the polyhedrin gene was not essential for the production of infectious virus and that deletion of certain sequences within the gene did not alter the control, or decrease the level of expression, of polyhedrin. An early viral protein of 25,000 molecular weight was apparently not essential for virus replication in vitro, as the synthesis of this protein was not detected in cells infected with a mutant virus.

Molecular Engineering of the Autographa californica Nuclear Polyhedrosis Virus Genome: Deletion Mutations Within the Polyhedrin Gene

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses.

In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several protein-based human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth ‘anniversary’ of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements.

Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology.

Summary The nuclear polyhedrosis viruses of Trichoplusia ni and Autographa californica produce plaques in monolayers of Spodoptera frugiperda cells under a solid overlay containing agarose as the solidifying agent. Plaques are visible macroscopically after staining the cells with neutral red or with the tetrazolium salt, INT, and a linear dose response is observed. The sensitivity of the assay is less than that obtained using an end-point dilution technique; however, plaque formation does provide a simple means of cloning virus.

A Plaque Assay for Nuclear Polyhedrosis Viruses using a Solid Overlay

Temperature-sensitive (ts) mutants were isolated from the baculovirus Autographa californica (alfalfa looper) MNPV, grown in Spodoptera frugiperda (fall armyworm) cells in the presence of N-methyl-N'-nitro-N-nitrosoguanidine. Of 567 plaque isolates screened, 27 were temperature sensitive (ts), representing a mutation frequency of 4.8%. Ten ts mutants were studied in detail: six failed to yield nonoccluded virus at 33 degrees C (NOV mutants), whereas the other four produced nonoccluded virus but were restricted in formation of polyhedra at 33 degrees C (Poly mutants). One of the six NOV mutants failed to synthesize viral DNA. Reversion and leak frequencies were determined, and the mutants were assorted into complementation groups based on the yield of polyhedrin synthesis in cells coinfected with pairs of mutants at 33 degrees C, as measured by radioimmunoassay. For NOV mutants, complementation indexes were also based on virus yield and were consistent with those based on polyhedrin synthesis. Nine mutants were assorted into five complementation groups. One mutant remained unclassified.

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups.

Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus

The nuclear polyhedrosis virus of Autographa californica has been titrated in Spodoptera frugiperda cells by the plaque method, using a solid overlay which does not require either the use of modified culture medium or expensive purified agarose or the addition of culture medium as a liquid layer above the solid agarose. This assay is more sensitive than that using a viscous methyl cellulose overlay but less sensitive than the end-point dilution technique. Neither Trichoplusia ni nor Bombyx mori cells were satisfactory as indicators for the assay as described, since they failed to form a stable monolayer. Manduca sexta cells could be utilized for assay of A. californica nuclear polyhedrosis virus, but the sensitivity was lower than with S. frugiperda cells.

Plaque assay of nuclear polyhedrosis viruses in cell culture.

Summary
Two morphology mutants designated m-5 and m-6, of Autographa californica nuclear polyhedrosis virus (NPV) were isolated from virus grown in the presence of N-methyl-N′-nitro-N-nitrosoguanidine. Infected cells contained single, large cuboidal inclusion bodies with a crystalline lattice ultrastructure and envelope similar to that of wild-type (wt) polyhedra. The inclusion bodies had low infectivity when fed to Trichoplusia ni larvae. The paracrystalline lattice structure of the mutants was similar to wt, but occluded viruses were rarely found within the mutants. The m-5 polyhedrin (mol. wt. 30000) could be distinguished from wt and m-6 polyhedrin on the basis of migration in SDS-PAGE gels. Peptide maps of polyhedrin were obtained following digestion with chymotrypsinogen or Staphylococcus aureus V8 protease. They were identical for m-6 and wt polyhedrin but m-5 polyhedrin gave a different pattern. Thus, the altered morphology may be due to a change in polyhedrin composition not detectable in m-6 polyhedrin by the methods used here, or it may be the result of a mutation affecting a protein not yet identified.

Martha Brown

Papers

A Plaque Assay for Nuclear Polyhedrosis Viruses using a Solid Overlay

Genetic Analysis of a Baculovirus, Autographa californica Nuclear Polyhedrosis Virus I. Isolation of Temperature-Sensitive Mutants and Assortment into Complementation Groups.

Factors affecting the yield of virus in a cloned cell line of Trichoplusia ni infected with a nuclear polyhedrosis virus

Plaque assay of nuclear polyhedrosis viruses in cell culture.

Characterization of Two Morphology Mutants of Autographa californica Nuclear Polyhedrosis Virus with Large Cuboidal Inclusion Bodies