Completion of RNA synthesis by viral RNA replicases

How the 5'-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA-DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5'-terminus and the C2'-hydroxyl of a ribose at the third nucleotide from the 5'-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.