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Abstract: Ebola virus (EBOV) causes severe hemorrhagic fevers in humans and non-human primates. While the role of the EBOV major matrix protein VP40 in morphogenesis is well understood, nothing is known about its contributions to the regulation of viral genome replication and/or transcription. Similarly, while it was reported that the minor matrix protein VP24 impairs viral genome replication, it remains unclear whether it also regulates transcription, since all common experimental systems measure the combined products of replication and transcription. We have developed systems that allow the independent monitoring of viral transcription and replication, based on qRT-PCR and a replication-deficient minigenome. Using these systems we show that VP24 regulates not only viral genome replication, but also transcription. Further, we show for the first time that VP40 is also involved in regulating these processes. These functions are conserved among EBOV species and, in the case of VP40, independent of its budding or RNA-binding functions. [Copyright &y& Elsevier]