Ebolaviruses (EBOV) belong to the group of nonsegmented negative-sense (NNS) RNA viruses and are highly pathogenic in humans. Currently, there are no approved therapeutics to treat or prevent EBOV hemorrhagic fever. Ebolaviruses use an RNA-dependent RNA polymerase to replicate and transcribe their genomes. Little is known about the molecular mechanisms employed by the EBOV polymerase to interact with the RNA template and initiate replication. Viral polymerases are common targets for antiviral drug development, and rational design of antiviral compounds targeting viral polymerases requires a thorough understanding of the molecular events involved in genome replication. Here, we propose to dissect the molecular mechanisms used by EBOV to initiate replication. Our preliminary data suggest that EBOV has evolved a replication initiation mechanism that is different from those used by other NNS RNA viruses. According to our model, EBOV initiates replication at position +2 of the template strand and uses an RNA secondary structure adopted by the promoter region to maintain its genome ends. To test this model, we will use RNA isolated from EBOV-infected cells or virions to determine the 3' and 5' ends of the replication products. By mapping the terminal nucleotides of intracellular and virion-associated viral RNA, we will identify the replication start sites. We will further use EBOV minigenome systems to introduce mutations in relevant regions and analyze the effects on replication initiation and genome integrity. We will perform the initial experiments using the EBOV prototype Zaire ebolavirus. We will then analyze if the identified replication mechanism used by ZEBOV is conserved among other EBOV species. Finally, we will determine the role of an RNA secondary structure adopted by the EBOV promoter region for promoter function and replication initiation. Overall, the proposed work will elucidate if the EBOV polymerase has acquired unique capabilities to interact with the viral genome or if it follows a general mechanism shared with other NNS RNA viruses.
The research proposed here will add to our knowledge of a group of viruses that includes several significant human pathogens. A better understanding of the differences and similarities in the viral life cycle of each virus family is key to the development of rationally designed antiviral drugs.
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