ANTIVIRAL ACTIVITY OF RNA MOLECULES CONTAINING SELF-RELEASING RIBOZYMES TARGETED TO LYMPHOCYTIC CHORIOMENINGITIS VIRUS

Ribozymes catalytically cleave substrate RNA molecules in a sequence-s pecific manner. Engineered ribozymes can be developed and introduced i nto tissue culture cells to regulate gene expression and to inhibit vi ral replication. We have previously reported on the construction of ce ll lines that constitutively express a single antiviral ribozyme embed ded in a lengthy RNA transcript. These cells exhibited a marked reduct ion in their ability to support viral infection. Here we report the co nstruction of RNA molecules that contain one or two antiviral ribozyme s, each specific for a different cleavage site on the genome of the ta rget virus, lymphocytic choriomeningitis virus (LCMV), and each contai ned in a self-cleavage cassette comprising cis-acting ribozymes design ed to release the antiviral molecules from the transcript. In vitro st udies showed that both antiviral ribozymes were released properly from the RNAs following cleavage by the flanking ribozymes and that these released ribozymes functioned as expected in cleaving the target virus RNA. These self-cleaving cassettes have been cloned into a retroviral vector downstream of, but in the same transcript as, the chlorampheni col acetyltransferase (CAT) gene. Thus, we hoped to employ CAT as a su rrogate marker of ribozyme transcription. Stably transformed cell line s were established. Cleavage by the cis-acting ribozymes was incomplet e, as assessed by Northern blot analysis and by the ability of transfo rmed cells to produce infectious retroviral particles. Nevertheless, t he antiviral ribozyme sequences exerted effects in tissue culture. LCM V RNA levels in ribozyme-expressing cells were suppressed, and infecti ous virus yields were decreased by up to 95% compared with normal cell s and with cells expressing inverted ribozymes. The antiviral effects correlated with CAT levels, but there was no significant difference be tween cell lines expressing a single ribozyme and those expressing two .