A study was undertaken to evaluate the efficacy of an adenoviral construct expressing the murine interferon-beta (IFN-beta) transgene (Ad:IFN-beta) against herpes simplex virus type 1 (HSV-1) infection in a primary trigeminal ganglion (TG) cell culture. The transduction efficiency ranged from 0.2 to 11.0% depending on the multiplicity of infection (m.o.i.) of the adenoviral vector (0.5-50.0). Moreover, neurons were the main target of the adenoviral transduction. TG cultures transduced with Ad:IFN-beta displayed up to a 19-fold reduction in viral titers compared with cells transduced with an Ad:Null or nontransduced TG culture controls. Transduction with Ad:IFN-beta up-regulated two critical antiviral genes, double-stranded RNA-dependent protein kinase R (PKR) and 2',5'-oligoadenylate synthetase (OAS). The absence of PKR or RNase L (downstream effector molecule of OAS) attenuated Ad:IFN-beta efficacy against HSV-1 replication, implicating a critical role for PKR and OAS/RNase systems in the establishment of IFN-induced resistance against HSV-1 in TG cells.