Inhibition of coronavirus replication by a ribozyme expressed from a defective interfering RNA

Colocalization of a therapeutic ribozyme with its viral target is necessary for virus inactivation but is often difficult to achieve. In this study, a hammerhead ribozyme was designed to cleave the linear (loop II) region of the frame-shifting pseudoknot in the RNA polymerase gene of the bovine coronavirus. When the 45-nt polymerase-targeted ribozyme sequence was cloned into the genome of a viral defective interfering RNA of the bovine coronavirus, no replication of transfected replicon RNA was observed in helper virus-infected cells. Under these conditions helper virus RNA accumulation, indicative of virus replication, was inhibited by greater than 50%. In its inactive form, the ribozyme caused no inhibition of DI RNA or helper virus replication after being transfected as a DI RNA transcript into helper virus- infected cells. These results indicate that the ribozyme is effective in trans against the polymerase gene and that the ribozyme must have colocalized with its target. The results suggest further that delivering an antiviral ribozyme from a defective RNA may be a generally useful strategy for inhibition of cytoplasmically replicating RNA viruses. Because of its suicidal nature, the ribozyme-containing replicon could in principle be used to cure persistent viral infection and leave the host cell unharmed. Such a therapeutic strategy might be desirable in chronic debilitating diseases in which persisting virus is a component of pathogenesis.