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Abstract

Orthoflaviviruses, such as Zika virus (ZIKV), are positive-sense RNA viruses responsible for a significant global disease burden, yet our understanding of their viral life cycle remains limited. Notably, the roles of several of the viral nonstructural proteins are still poorly understood, limiting the development of targeted therapeutics. The orthoflaviviral nonstructural protein 2A (NS2A) is involved in both viral RNA replication and virion assembly; nevertheless, the mechanisms by which NS2A mediates these functions remain unclear. Interestingly, the protein is known to exist in several isoforms during infection, including both a smaller, truncated isoform, termed NS2Aɑ, as well as several higher molecular weight forms; however, there is a limited understanding of how the various forms of NS2A contribute to its distinct roles in the viral life cycle. Although ZIKV NS2A has been presumed to oligomerize, the oligomerization interface has not been determined to date. Thus, we sought herein to further explore the higher molecular weight forms of NS2A present during infection by investigating both NS2A oligomerization and post-translational modifications (PTMs). By conducting co-IP assays with NS2A truncation and domain deletion mutants, we mapped the ZIKV NS2A oligomerization interface to the N-terminus (amino acids 1–73), which is located in the endoplasmic reticulum (ER). Specifically, we were able to implicate the first ER amphipathic helix (amino acids 6-31) and the ER linker region (amino acids 56-73) in NS2A oligomerization; however, to date, we have been unable to confirm the involvement of residues 32-55. Additionally, through mass spectrometry analysis, we identified two ubiquitin (Ub) or Ub-like protein (UBL) modifications on residues K186 and K193 in the C-terminus of ZIKV NS2A during infection. The introduction of lysine to arginine (K-to-R) mutations in this region resulted in a significant decrease in infectious particle production, but did not prevent NS2Aɑ biogenesis or impact viral RNA replication. Collectively, this work expands our knowledge on the ZIKV NS2A protein by identifying the domain(s) of NS2A that contribute to oligomerization and also highlights the critical role of NS2A PTMs in virion assembly.