Zika virus produces two isoforms of the NS2A protein that play mutually exclusive roles in viral replication and virion assembly.

Zika virus (ZIKV), dengue virus, and several other orthoflaviviruses cause disease outbreaks in human and animal populations worldwide. The orthoflaviviral nonstructural protein 2A (NS2A) is critical for both viral replication and virion assembly. However, due to its small size, hydrophobicity, and the absence of structural information or protein-specific antibodies, we lack a clear understanding of how NS2A participates in these disparate processes. Here, using endogenously tagged NS2A-containing viral RNAs, we reveal that two NS2A isoforms are present during ZIKV infection, including both full-length NS2A and a C-terminally truncated isoform, termed NS2Aα. Phylogenetic and conservation analyses combined with polyprotein cleavage assays suggest that both NS2A isoforms are generated via the viral protease and are conserved across the Orthoflavivirus genus. Finally, using assays for viral replication and infectious particle production, we demonstrate that the two distinct NS2A isoforms function at different stages of the viral life cycle, with full-length NS2A required for viral RNA replication and NS2Aα important for virion assembly. Taken together, our data helps shed light on the form(s) and function(s) of the NS2A protein and suggests that each NS2A isoform plays a mutually exclusive role in the orthoflaviviral life cycle.