Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidyl inositol (GPI) membrane anchor in neurons and polarised epithelial cells

Abstract

The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosylphosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell–cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.