Functional characterization of cellular RNA-binding proteins that support Flavivirus replication

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An essential prerequisite for the replication of Flaviviruses is the cyclization of the viral positive-strand RNA genome, which enables the virus-encoded replicase to initiate RNA synthesis. During earlier studies, we defined the p45 isoform of the AU-rich element RNA-binding protein 1 (AUF1) as a cellular host factor that considerably supports the RNA replication process of the mosquito borne Flavivirus West Nile virus (WNV) in human cells. A set of complementary experimental data indicated that AUF1 p45 has an RNA chaperone activity, which supports RNA cyclization and viral RNA synthesis by destabilizing a stem-structure at the viral RNA's 3'end. Moreover, the protein was shown having an RNA annealing activity, which is suspected to assist the hybridization of complementary cyclization sequences in the 5'- and 3'regions of the viral genome. The proposed studies of this application aim at gaining a further detailed picture on the activities of AUF1 p45 and of potential mosquito analogues of the protein while operating as host factors on flaviviral RNAs. Three interrelated questions will be addressed: (1) What are the molecular determinants on the side of the AUF1 p45 protein and on the side of the flaviviral RNA that enable the protein to destabilize the viral RNA on the one and to support RNA annealing on the other hand? (2) Does AUF1 p45 act in a similar way with other flaviviruses, e.g. the WNV-related Dengue virus (DENV)? (3) Do mosquito cells encode similar types of host factors that have homologous functions as the human AUF1 p45? The proposed project is expected to generate new knowledge in two respects. On the one hand, we will obtain additional insights into the molecular mechanisms on how a cellular RNA-binding protein (RBP) is capable to modulate the activity of viral replication machineries. Moreover, we anticipate gaining important information on the general mechanisms of how RBPs / RNA chaperones control the structures and functions of RNA molecules.