A gene delivery/recall system for neurons which utilizes ribonucleotide reductase-negative herpes simplex viruses

Abstract

We present data which show that ribonucleotide (RR)-negative herpes simplex virus type-1 (HSV-1) is a useful vector for gene delivery into neuronal cells. For these studies we used hrR3, a genetically engineered HSV-1 mutant which has an in-frame insertion of the bacterial lacZ gene into the HSV gene that encodes the large subunit (ICP6) of RR. After infection of rat primary sympathetic neuronal cultures with hrR3, the ICP6:: lacZ chimeric gene was expressed, as shown by blue staining of the cells upon exposure to X-Gal, a chromogenic β-galactosidase substrate. When the infection was performed in the presence of acyclovir, hrR3 appeared to become “latent”; neither infectious virus nor β-galactosidase activity was detectable in these neuronal cultures at 3 weeks after the acyclovir was removed. However, β-galactosidase activity was inducible in the “latent” cultures by superinfection with ICP6Δ (a RR-negative deletion mutant) without resulting in the “reactivation” of hrR3 and without apparent cytopathic effects. In contrast, superinfection with ICP6Δ + 3.1, a virus derived by marker rescue of ICP6Δ, resulted in the expression of lacZ, the release of hrR3 into the culture medium, and cytopathic effects. The introduction of a foreign gene into neuronal cells by a RR-negative herpes simplex virus, and the subsequent induction of gene expression by another noncomplementing virus, may constitute a prototype gene delivery/recall system for neurons.