The DA strain of Theiler's murine encephalomyelitis virus (TMEV), a member of the Cardiovirus genus of the family Picornaviridae, causes persistent infection in susceptible mice, associated with restricted expression of viral proteins, and induces a demyelinating disease of the central nervous system. DA-induced demyelinating disease serves as a model of human multiple sclerosis because of similarities in pathology and because host immune responses contribute to pathogenesis in both disorders. In contrast, the GDVII strain of TMEV causes acute lethal encephalitis with no virus persistence. Cardiovirus L is a multifunctional protein that blocks beta interferon (IFN-beta) gene transcription. We show that both DA L and GDVII L disrupt IFN-beta gene transcription induction by IFN regulatory factor 3 (IRF-3) but do so at different points in the signaling pathway. DA L blocks IFN-beta gene transcription downstream of mitochondrial antiviral signaling protein (MAVS) but upstream of IRF-3 activation, while GDVII L acts downstream of IRF-3 activation. Both DA L and GDVII L block IFN-beta gene transcription in infected mice; however, IFN-beta mRNA is expressed at low levels in the central nervous systems of mice persistently infected with DA. The particular level of IFN-beta mRNA expression set by DA L as well as other factors in the IRF-3 pathway may play a role in virus persistence, inflammation, and the restricted expression of viral proteins during the late stage of demyelinating disease.