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 polymerase of disrupted (open) cores is able to catalyze the synthesis of dsRNA from exogenous viral mRNAs in vitro. In this study, we have identified sequences 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 addition of oligonucleotides which were complementary to nonconserved sequences present 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 replication. The fact that antisense oligonucleotides inhibited dsRNA synthesis indicates that the strandedness (single- versus double-stranded) and secondary structure of the viral mRNA template are factors that affect the efficiency of minus strand synthesis.