Increasing baculovirus transduction of mammalian cells for drug discovery

The use of the insect virus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a gene delivery vector for mammalian cells began in 1995 with the discovery that mammalian hepatocytes could be efficiently infected by the virus (40). Since then, its use has increased steadily as a result of the number of advantages the system confers: an inability to replicate in mammalian cells or cause cellular toxicity, efficient entry in certain cell lines, inherent flexibility of use and additional benefits specific to the transient expression of drug targets. However, the system has retained the limitation of inefficient entry into some mammalian cells, limiting the usefulness of the system to particular cell lines. To improve the breadth of mammalian cell entry exhibted by AcMNPV the mechanisms of promiscuous cell entry were investigated. Initial results revealed that simple physical and chemical techniques such as using low pH diluents, optimising transduction volumes and increasing access to the mammalian plasma membrane could be applied to target cell populations to increase their susceptibility to entry by AcMNPV, and also provided supporting evidence for later experiments on the mechanism of cell entry. Focussing on the AcMNPV envelope protein gp64, genetic mutations introduced into the basic loop region of the protein between residues 268 and 281 was shown to be important for virus entry. In particular, the addition of basic residues to the crown of the basic loop increased entry into mammalian cells and resulted in an inability to transmit between adjacent insect cells. Structural modelling of the pre-fusion form of gp64 based on the available crystal structure for the VSVG fusion protein was consistent with the view that a role for the basic loop may be in the initial step of receptor-independent cell contact. The charge on gp64, principally associated with the loop, may interact with charged moieties on the cell plasma membrane, followed by structural rearrangements of gp64 that induce viral entry and fusion at the cell surface or following uptake into an endosome. Further experimentation revealed that entry efficiency is not solely dependent on the fusion activity of gp64 but that membrane lipid composition also impacts the fusion process between the insect virus and mammalian cells. 4EThOS - Electronic Theses Online ServiceGBUnited Kingdo