Mechanism of Viral RNA-dependent RNA Synthesis

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Replication and transcription from RNA templates are required for RNA viruses to cause diseases. These processes are also mechanistically interesting since the viral replication enzymes are the only class of template-dependent polymerases that naturally initiate and terminate RNA synthesis from the termini of the templates. Viral replication enzymes, hereafter called the replicase, are generally composed of a virally encoded RNA-dependent RNA polymerase (RdRp), and additional viral and host subunits. The replicase from Brome mosaic virus (BMV) can be enriched and solubilized from infected plants, thus permitting biochemical characterization. The BMV replicase can specifically recognize BMV RNAs and then precede through a number of biochemical steps, including de novo initiation, elongation, termination, and RNA recombination. Requirements for these steps in vitro nicely mimic the requirements for replication and recombination in vivo, thus providing insight into the least understood class of template-dependent polymerases. The goal of this project is to better define how the BMV replicase recognizes specific RNA sequences and goes through different steps of RNA synthesis.This research has four specific aims: 1) to examine the interaction between the replicase and core promoter RNAs; 2) to identify and characterize RNA sequences that cause pausing of the elongating replicase complex during RNA synthesis; 3) to examine the mechanism of RNA recombination in vitro; and 4) to further purify and characterize the components in the BMV replicase. Knowledge gained from the execution of these objectives will contribute to the basic understanding of virus replication. The use of BMV as a model system will directly benefit less well-studied systems that can cause diseases such as encephalitis, measles, and hepatitis. The detailed understanding of the different steps in viral RNA replication should uncover targets for the design of highly specific antiviral drugs.