RP02 Project Summary/Abstract for Contributions of Ebola and Marburg virus VP30 and VP24 proteins to viral RNA synthesis, assembly and egress Marburg virus (MARV) and Ebola virus (EBOV) are deadly emerging viruses of the filovirus family. Major gaps remain in our understanding as to how filoviral-filoviral and filoviral-host interactions promote replication and severe disease. Contributing to these limitations in knowledge is the requirement to study replicating filoviruses under biosafety level 4 (BSL4) containment. To overcome the BSL4 limitation, we have produced replication cycle modeling assays, called transcription-replication competent virus-like particle (trVLP) assays, that recapitulate each of the major steps in the EBOV and MARV replication cycles at BSL2. These tools enable the efficient analysis of filoviral and host proteins in the context of a replicating system. These systems also permit the study of manipulations such as lethal mutations or elimination of key host factors that are not compatible with replication of live virus. With these assays, we have explored the role of MARV VP30 (mVP30) and MARV VP24 (mVP24) that exhibit distinct functional properties compared to their EBOV homologues. EBOV VP30 is required for transcription initiation of the EBOV nucleoprotein (eNP) gene and is regulated by host proteins with PPxPxY motifs. In contrast, mVP30 is reported to not be required for viral transcription in minigenome assays, yet it is essential for virus growth. Why it is essential has been unclear. EBOV VP24 binds importin alpha proteins to block interferon signaling and also participates in the maturation of nucleocapsid formation, modulates viral RNA synthesis, and is critical for filoviral infectivity. In contrast, mVP24 interacts with host protein Keap1, a regulator of cellular antioxidant responses. Our MARV trVLP data suggests that mVP30 is essential for efficient production of viable virus, due to its requirement for transcription of the mGP gene, and that mutations disrupting mVP24-Keap1 interaction substantially decrease infectivity. Given these findings, we will test the hypothesis that mVP30 plays a critical role in mGP transcription initiation; we will characterize secondary structures that we hypothesize confer mVP30 dependence on the mGP gene; and we will evaluate how interacting host proteins modulate mVP30 function. We will also test the hypothesis that mVP24 interaction with Keap1 is required for viral growth and define specific replication steps for which mVP24-Keap1 interaction is required. Finally, with our trVLP assays, we will define the mechanisms by which other filovirus- host interactions identified in Research Projects 1 and 3 affect virus growth. Together, these studies will substantially enhance the understanding of interactions that play key roles in filovirus replication and pathogenesis.