Recombinant Alphavirus-Mediated Expression of Ion Channels and Receptors in the Brain

Abstract

AbstractEctopic gene transfer has been a crucial method for neurobiological studies aiming to resolve the function of specific membrane and non-membrane proteins. For this purpose, expression vectors based on Semliki Forest virus, Sindbis virus, and Venezuelan equine encephalitis virus, all members of the Alphavirus genus, have been engineered in the form of naked RNA replicons, recombinant viral particles, and layered DNA vectors. By applying these alphavirus vectors to mammalian cell lines, large quantities of integral membrane proteins including ligand- and voltage-gated ion channels as well as G protein-coupled receptors have been expressed, and their functional activity has been confirmed by both electrophysiological recordings and coupling to G proteins. Furthermore, alphavirus vectors have provided efficient delivery tools for high-level expression of recombinant proteins in cultures of primary neurons and organotypic hippocampal slices, as well as upon intracranial injection, in vivo in rodent brain. This chapter describes the generation of infectious but replication-deficient Semliki Forest virus and Sindbis virus particles that are useful for the efficient transduction of neurons in vitro, in situ, and in vivo.Key wordsSemliki Forest virus Sindbis virus Recombinant proteinVoltage-gated sodium channelG protein-coupled receptorCell linePrimary neuron Hippocampal sliceRodent