Subcellular determinants of orthoflavivirus protease activity

Orthoflaviviruses are small, enveloped, positive-sense RNA viruses that cause over 500 million infections globally each year for which there are no antiviral treatments. The viral protease is an attractive target for therapeutics due to its critical functions throughout infection. Many studies have reported on the structure, function, and importance of orthoflavivirus proteases; However, the molecular determinants for cleavage of intracellular substrates by orthoflavivirus proteases and how these factors affect viral fitness are unknown. In this study, we used our fluorescent, protease-activity reporter system to investigate the subcellular determinants involved in orthoflavivirus protease cleavage. By modifying our reporter platform, we identified endoplasmic reticulum (ER) subdomain localization and membrane proximity of the substrate cut site as two previously uncharacterized molecular determinants for cleavage. We also altered the amino acid composition of the reporter cut site to introduce sequences present at the cytoplasmic junctions within orthoflavivirus polyproteins and found that each protease processed the sequence located at the junction between NS4A and the 2K peptide least efficiently. Live-cell imaging revealed that cleavage of the NS4A|2K sequence is significantly delayed compared to the capsid cleavage sequence. We further determined that introducing a more efficient cleavage sequence into the NS4A|2K junctions of orthoflavivirus infectious clones abolished virus recovery. Overall, this study identifies ER subdomain localization and membrane proximity of the cut site as molecular determinants for cleavage by orthoflavivirus proteases and provides insight into the role that sequence specificity plays in the coordinated processing of the viral polyprotein and establishing productive infections. ImportanceOrthoflaviviruses are the most prevalent and dangerous arthropod-borne viruses (arboviruses) leading to over 500 million global infections annually. Orthoflavivirus infection can cause severe pathologies, including hemorrhagic conditions and neurological disease, that lead to hundreds of thousands of deaths each year. The viral protease complex, responsible for processing the viral polyprotein into its functional subunits, is an attractive target for antiviral therapeutic development. Despite extensive research efforts on these viral protein complexes, all protease inhibitor candidates have fallen short of clinical efficacy, highlighting a considerable gap in knowledge of the viral proteases complex intracellular activity. The significance of our research is in characterizing the subcellular determinants associated with orthoflavivirus protease cleavage efficiency and how these factors can influence viral fitness. These findings contribute to closing this gap in knowledge of the mechanisms of orthoflavivirus proteases which can ultimately lead to the successful development of targeted antivirals.