Marburg virus (MARV) belongs to the filovirus family and is highly pathogenic in humans. Despite being classified as Category A Priority Pathogen by NIAID, and its potential to cause large-scale outbreaks, similar to the recent Ebola virus outbreak, research on MARV lags significantly behind that on other non-segmented negative sense (NNS) RNA viruses. Here, we propose to perform in-depth analyses of MARV transcription and gene expression. Dissecting the mechanisms of MARV gene expression will not only be instrumental for the targeted development of antiviral drugs, it will also reveal unifying paradigms and distinctions between the NNS RNA viruses. The filovirus genome is transcribed by a virally encoded RNA-dependent RNA polymerase complex, which is capable of generating capped and polyadenylated mRNAs. This process occurs in the cell cytoplasm, close to ribosomes and cellular RNA binding proteins. This project will examine three different stages of MARV gene expression. In Aim 1, we will elucidate the mechanism of transcription initiation at the MARV promoter and investigate the role of structural features of the polymerase in this process. Notably, the MARV promoter sequence has some unusual features, and we intend to explore the functional relevance of these characteristics. In Aim 2, we will determine the function of conserved hairpin loops that are formed at the 5´ end of each MARV mRNA. We will explore the effect of these structures on transcription, RNA stability, trafficking and translation. In Aim 3, we will focus on mRNA polyadenylation and release. The mechanism of mRNA release is not well understood for any NNS RNA virus, and the results obtained in this aim will help to broaden our understanding of NNS RNA virus transcription strategies. This proposal brings together expertise in studying NNS RNA polymerases, MARV molecular biology, and mRNA-protein interactions. Together, the research team has established a unique tool set to achieve the goals of this proposal, including a MARV in vitro polymerase assay, various MARV reverse genetics systems, and highly innovative single molecule mRNA-protein binding assays. These studies will shine new light on a crucial aspect of MARV infection and enhance our understanding of NNS RNA virus biology.