Manipulation of baculovirus vectors.

Baculovirus expression vectors provide an excellent system for the synthesis of recombinant proteins in insect cells. This article presents sufficient background information to allow the nonspecialist to understand the basic principles of the technology and the development of baculovirus expression vectors. A summary of the most commonly used plasmids and viruses is presented. Detailed techniques are described to enable recombinant baculoviruses to be constructed. These methods include the protocols required for propagating insect cells in culture and their subsequent infection with viruses.

An Autographa californica nucleopolyhedrovirus lef-2 mutant: consequences for DNA replication and very late gene expression.

In order to define factors involved in very late Autographa californica nucleopolyhedrovirus (AcMNPV) gene function, random mutagenesis of a baculovirus recombinant (AcUW1.lacZ) by 5'-bromodeoxyuridine treatment was performed. Five viruses were selected with deficiencies in very late gene expression. These were characterized by complementation analysis. One mutant virus, VLD1, was found to be completely deficient in very late gene function. This virus could be complemented by a helper virus to express the very late genes, suggesting that the mutant virus was defective in an activator of very late gene expression. Further studies revealed that the replication of VLD1 was temporally delayed when compared to wild-type virus. The mutation in VLD1 was mapped to a subfragment of the EcoRI-I region of the AcMNPV genome between 0 and 5 map units. Sequence analysis revealed the presence of point mutations in ORF2 and in lef-2. Further mapping experiments demonstrated that only replacement of the point mutation in lef-2 with a wild-type sequence could restore VLD1 to a normal phenotype. Previous studies have suggested that the lef-2 gene product is involved in DNA replication. This was investigated by comparison of DNA replication in wild-type- and VLD1-infected cells. It was found that the mutation in the lef-2 gene of VLD1 did not have an effect on DNA replication. It is proposed that lef-2 may play a dual role, both in DNA replication and very late gene expression.