Elucidating the Proteolytic Determinants of Flavivirus Infection

Flaviviruses, recently renamed to orthoflavivirus, are small, enveloped viruses with positive-sense, single-stranded RNA (ssRNA) genomes of about 11 kilobases. Upon cell entry and uncoating, the viral RNA (vRNA) is directly translated at the endoplasmic reticulum (ER) as a single polyprotein that is then processed by the viral and cellular proteases into 10 functional subunits, consisting of three structural proteins and seven nonstructural (NS) proteins. Among the NS proteins are NS2B and NS3, which form the viral protease complex. NS3 consists of a serine protease domain while NS2B is anchored to the ER and contains a cytoplasmic loop that serves as a cofactor required for the catalytic activity of the protease. The NS2B3 protease 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 viral protease; however, the molecular determinants for flavivirus protease cleavage of intracellular substrates and how these factors affect viral fitness are unknown. In the first half of this work, we developed and validated a reporter platform that detects intracellular flavivirus protease activity. By introducing various modifications into our tractable reporter system, we identified two previously uncharacterized molecular determinants associated with flavivirus protease cleavage efficiency. Specifically, we found that the ER membrane proximity of the protease recognition motif of the substrate as well as its localization to distinct subdomains of the ER greatly affect cleavage efficiency by flavivirus proteases. To our knowledge, this work is the first characterization of molecular determinants for flavivirus protease activity outside of primary sequence specificity. In the second half of this work, we introduced relevant primary sequences into our reporter construct and found that each flavivirus protease processed the motif located at the NS4A|2K junction of the polyprotein with poor cleavage efficiency. Using our reporter system with live-cell imaging to investigate comparative cleavage kinetics, we observed a significant delay in the processing of the NS4A|2K junction motif during dengue virus (DENV) infection. Further, we found that increasing the rate of cleavage at the NS4A|2K junction within flavivirus infectious clones was deleterious to viral fitness. Subsequent experiments revealed that this detriment was attributed to the inhibition of viral replication possibly due to a restriction in replication organelle formation upon premature cleavage at the NS4A|2K junction. Collectively, these findings characterize the molecular determinants of flavivirus protease activity and uncover the impact of cleavage specificity on infection.