Successful Rescue of Synthetic AcMNPV with a ~17 kb Deletion in the C1 Region of the Genome

Abstract

Baculoviruses have been widely used as expression vectors. However, numerous genes in the baculoviral genome are non-essential for cellular infection and protein expression, making the optimisation of baculovirus expression vectors possible. We used a synthetic biological method to reduce the number of genes in a partial region of the autograph californica multiple nucleopolyhedrovirus (AcMNPV), the most widely used baculovirus expression vector. The C1 region of the AcMNPV is 46.4 kb and is subdivided into B1, B2, and B3 fragments. We first designed modified B1, B2, and B3 fragments by deleting the non-essential genes, and then synthesised complete viral genomes containing either individual modified B fragments or joint modified B fragments through transformation-related recombination in yeast. The synthetic genomes were then transfected into Sf9 cells to rescue the progeny viruses and test their infectivity. The design-build-test cycle was repeated until the ultimately rescued virus could produce progeny viruses efficiently. Finally, AcMNPV-Syn-mC1-1.1 by deleting approximately 17.2 kb, including 20 ORFs, in the C1 region, was obtained. This is essential to the synthesis of a minimal AcMNPV genome that can generate infectious progeny viruses and can be further used to optimise the foundation of baculovirus expression vectors.