ANALYSIS OF VERY LATE GENE-EXPRESSION BY AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS AND THE FURTHER DEVELOPMENT OF MULTIPLE EXPRESSION VECTORS

The consequences of locating the polyhedrin gene coding sequences and the p10 promoter at heterologous positions within the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome were investigated. Positioning the polyhedrin or β-galactosidase coding sequences under the control of the p10 gene promoter via the use of the new transfer vector, pAcUW1, resulted in viable recombinant viruses able to produce high levels of each non-fused gene product at the appropriate time. Polyhedra were also produced by the virus with the p10 promoter-polyhedrin hybrid gene and appeared normal in thin sections. Therefore the combination of polyhedrin promoter and coding sequences is evidently not essential for efficient expression of this protein. The p10 promoter can serve this function equally well. Viruses with the p10 promoter and β-galactosidase coding sequences placed upstream from the polyhedrin gene in either orientation produced large amounts of β-galactosidase protein in infected cells, thus demonstrating that the p10 promoter can function at an alternative position within the virus genome. A second transfer vector, pAc-UW2B, was constructed, with a copy of the p10 gene promoter placed upstream and in opposition to the polyhedrin gene. This mediates the insertion of any foreign gene under the control of the p10 promoter while preserving normal p10 gene expression. The advantages of these constructs over the conventional vectors presently used to express foreign genes in insect cell systems and their utilization in the production of virus insecticides are discussed.