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 molecular determinants of flavivirus
protease cleavage and the role that sequence specificity plays in infection. Understanding the infectious role of
flavivirus protease specificity will aid in the development of antiviral therapeutics targeting this viral protein.