Initiation of (−)-Strand RNA Synthesis Catalyzed by the BMV RNA-Dependent RNA Polymerase: Synthesis of Oligonucleotides

AbstractRNA replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. Partially purified RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley leaves is able to synthesize (−)-strand RNAs from input (+)-strand templates. In resolving RdRp products generated during (−)-strand BMV RNA synthesis, we found that an RNA of eight nucleotides was generated at approximately 10-fold molar excess to the full-length (−)-strand RNA. Production of the 8-mer was dependent upon and specific to BMV RNA templates. Furthermore, inhibitors of full-length (−)-strand RNA synthesis by RdRp also affected the production of the 8-mer. Analysis of the sequence of the 8-mer suggests that it is initiated at the authentic initiation site on the BMV RNA template, consistent with the possibility that the 8-mer is an abortive initiation product of RNA synthesis by RdRpin vitro.Addition of heparin or Mn2+differentially affected production of the 8-mer and full-length (−)-strand RNA, as did some nucleotide changes near the site of RNA initiation. These studies further define the initiation process of (−)-strand RNA synthesis