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A controlled mutational study was used to determine the site and mechanism of the antiviral action of ribozymes that inhibit Sindbis virus replication. A hairpin ribozyme targeting G575 of the Sindbis virus genomic RNA was designed and cloned into a minimized alphavirus amplicon vector. Cells that were stably transfected with this construct expressed low levels of a constitutive transcript containing the ribozyme plus recognition sequences for Sindbis RNA replicase. Upon infection, the ribozyme transcript was amplified to high levels by the viral replicase, resulting in decreased viral production from infected ribozyme-expressing cells. Mutations were then introduced into the viral RNA target sequence to interfere with ribozyme binding, and compensatory changes were generated in the ribozyme recognition sequence. Single mutations in the virus or ribozyme decreased the efficacy of the ribozyme's inhibition of viral replication, and compensatory mutations restored it. To confirm that ribozyme-catalyzed RNA cleavage was actually needed for inhibition, we performed tests with a cell line expressing an inactivated ribozyme and with a virus containing a single nucleotide target mutation that allowed the ribozyme to bind but blocked cleavage at the recognition site. The results show that most of the antiviral activity of ribozymes is due to ribozyme-catalyzed cleavage at the targeted RNA sequence, but some additional inhibition seems to occur through an antisense mechanism.

Inhibition of Viral Replication by Ribozyme: Mutational Analysis of the Site and Mechanism of Antiviral Activity