UFMylation promotes orthoflavivirus infectious particle production.

Post-translational modifications play crucial roles in regulating viral infections, yet roles for many modifications remain unexplored in orthoflavivirus biology. Here, we demonstrate that the UFMylation system, a post-translational modification pathway that catalyzes the transfer of UFM1 onto proteins and promotes infection by multiple orthoflaviviruses, including dengue virus (DENV), Zika virus (ZIKV), West Nile virus, and yellow fever virus. We found that depletion of the UFMylation E3 ligase complex proteins UFL1 and UFBP1, as well as other UFMylation machinery components (UBA5, UFC1, and UFM1), significantly reduces orthoflavivirus infectious virion production. This regulation was specific to orthoflaviviruses as the hepacivirus and member of the broader Flaviviridae family, hepatitis C virus, was not regulated by UFL1. Mechanistically, UFMylation did not regulate viral RNA translation, RNA replication, or virion egress but instead affected the assembly of infectious virions. Furthermore, we identified novel interactions between UFL1 and several viral proteins involved in orthoflavivirus virion assembly, including NS2A, NS2B-NS3, and capsid. These findings establish UFMylation as a previously unrecognized post-translational modification pathway that promotes orthoflavivirus infection through modulation of viral assembly. This work expands our understanding of the post-translational modifications that control orthoflavivirus infection and identifies new potential therapeutic targets.IMPORTANCEOrthoflaviviruses depend on host-mediated post-translational modifications to successfully complete their life cycle, yet many of these critical interactions remain undefined. Here, we describe a role for a post-translational modification pathway, UFMylation, in promoting infectious particle production of ZIKV and DENV. We show that UFMylation is dispensable for initial RNA translation and RNA replication but promotes the assembly of infectious virions. Additionally, we find that regulation of infection by UFMylation extends to other orthoflaviviruses, including West Nile virus and yellow fever virus, but not to the broader Flaviviridae family. Finally, we demonstrate that UFMylation machinery directly interacts with specific DENV and ZIKV proteins during infection. These studies reveal a previously unrecognized role for UFMylation in regulating orthoflavivirus infection.