Identification of sequences in rotavirus mRNAs important for minus strand synthesis using antisense oligonucleotides

The core of the rotavirion consists of three proteins, including the viral RNA polymerase, and 11 segments of double-stranded (ds)RNA. The RNA polymer ase of disrupted (open) cores Is able to catalyze the synthesis of dsRNA fr om exogenous viral mRNAs in vitro. In this study, we have identified sequen ces in exogenous viral mRNAs important for RNA replication using antisense oligonucleotides. The results showed that oligonucleotides complementary to the highly conserved 3'-terminal sequence of rotavirus mRNAs prevented all but basal levels of dsRNA synthesis. Notably, we observed that the additio n of oligonucleotides which were complementary to nonconserved sequences pr esent either at the 5'- or 3'-end of a viral mRNA effectively inhibited its replication without interfering with the replication of other viral mRNAs present in the same replication assay. Thus, the nonconserved sequences in rotavirus mRNAs contain gene-specific information that promotes RNA replica tion. The fact that antisense oligonucleotides Inhibited dsRNA synthesis in dicates that the strandedness (single- versus double-stranded) and secondar y structure of the viral mRNA template are factors that affect the efficien cy of minus strand synthesis. (C) 2001 Academic Press.