PROJECT SUMMARY Flaviviruses are small, enveloped viruses with positive-sense, single-stranded RNA genomes of about 11 kilobases. Upon cell entry and uncoating, the vRNA traffics to the endoplasmic reticulum (ER) where it is translated as a single polyprotein that is then post-translationally processed by the viral and cellular proteases into 10 functional subunits, consisting of three structural proteins and seven non-structural (NS) proteins. Among the NS proteins are NS2B and NS3. NS3 consists of a serine protease and a helicase domain while NS2B is anchored to the ER and has a cytoplasmic loop that serves as a cofactor required for the catalytic activity of the NS3 protease domain. The NS2B3 complex is responsible for all cytoplasmic cleavage events of the viral polyprotein, making it an essential protein complex with functions required for the viral lifecycle. Many studies have reported on the structure, function, and importance of the NS2B3 protease; However, the molecular determinants for flavivirus protease cleavage of intracellular substrates and how these factors affect viral fitness is unknown. Using an intracellular protease activity reporter developed by our laboratory, we found that a soluble form the reporter was not cleaved whereas an ER-anchored reporter was efficiently cleaved, suggesting there are multiple determining factors within a substrate required for protease cleavage. Aim 1 will test the hypothesis that there are other molecular determinants for flavivirus protease cleavage of substrates outside of primary cleavage sequence. This will be addressed through manipulation of the intracellular localization of the reporter substrate and the distance of the cleavage site from organelle membranes. Although the typical cleavage motif of substrates is known, there is still some variability in the primary sequences cleaved by the protease at different junctions within the viral polyprotein. We were interested in understanding the variability in cleavage efficiency between the different sequences present in the viral polyprotein junctions of DENV, ZIKV, WNV, and YFV. Our preliminary data showed that each flavivirus protease has its own unique cleavage profile and that each flavivirus protease tested processed the sequence located at the junction between NS4A and the 2K peptide of its polyprotein least efficiently. Further, we determined that introducing a more efficient cleavage site into the NS4A/2K junction of a DENV infectious clone leads to the complete loss of viral recovery. Aim 2 will test the hypothesis that aberrant cleavage at the NS4A/2K junction of the flavivirus polyprotein causes detrimental effects to viral fitness. Using multiple genetic tools, we will assess the effect of this mutation on different stages of the viral lifecycle, including replication, polyprotein stability/processing, and replication organelle formation. Together, the results produced from this proposal will advance our understanding of the molecul...