The importance of the effector functions performed by glial cells, the resident cells of the CNS possessing immune functions, in abrogating potentially fatal neurotropic viral infections have only recently begun to gain appreciation. These effector functions would not be possible if it weren't for the presence of innate pattern recognition receptors (PRRs) that serve as sensors for conserved pathogenic components and initiate activation of innate defense mechanisms in response to a non-self invader. The current study investigates the role of retinoic acid inducible gene (RIG)-I and DNA-dependent activator of interferon regulatory factors (DAI) in the recognition and intervention against RNA and DNA viral infections in the glial cells, astrocytes and microglia. Our laboratory has previously shown that astrocytes and microglia express RIG-I and DAI and demonstrated that stimulation of these receptors with synthetic or virally-derived RNA or DNA ligands, respectively, leads to the expression and secretion of proinflammatory mediators. More recently, it has been hypothesized that the utilization of RNA polymerase III confers on RIG-I the ability to indirectly recognize and become activated by DNA pathogens. In the present study, we demonstrate that RNA polymerase III inhibition significantly attenuates protective host responses elicited following challenge with DNA, but not RNA, viruses and ligands. Inhibition of RNA polymerase III activity led to enhanced DNA, but not RNA, viral replication associated with reduced production of the potent antiviral molecule, interferon-beta (IFN-beta), suggesting that RNA polymerase III licenses RIG-I to mount protective immune responses against DNA pathogens.