Coronaviruses are a family of RNA viruses that cause respiratory, gastrointestinal and neurological diseases in a variety of animals including humans. Coronavirus RNA synthesis is unusual because viral mRNAs are generated via a discontinuous mechanism during which a leader RNA and mRNA body sequence become contiguous. Furthermore, replicating viral RNAs undergo high frequency RNA recombination. To understand these unique mechanisms of viral RNA synthesis, we are characterizing the coronavirus replicase, the enzyme responsible for RNA synthesis and recombination. The corona virus replicase is synthesized as an 800 kDa polyprotein that is processed by viral proteinases. In this proposal, we will investigate how the replicase polyprotein is processed, assembled on intracellular membranes, and functions in viral RNA synthesis. We hypothesize that certain replicase products act as the scaffold for assembly of the replicase complex onto membranes, and that this assembly is essential for the generation of the functional replicase. Using a series of polyclonal antisera generated to individual replicase products, we will determine the subcellular localization of replicase products by confocal and immuno-electron microscopy. Biochemical methods and a system for the cytoplasmic expression of replicase products will be used to determine if individual replicase products act as integral membrane proteins or if their localization is dependent of other replication products. We will also use the cytoplasmic expression system to express replicase products in trans and determine if they can complement temperature sensitive mutants defective in viral RNA synthesis. These studies will provide new information of a viral replicase that mediates discontinuous mRNA synthesis and RNA recombination, events that contribute to the ability of viruses to rapidly evolve, evade the immune system and hamper vaccine development.
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