{ "abstract": "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. Author summary: The Orthoflavivirus NS2A protein is a small, hydrophobic, integral membrane protein implicated in both viral RNA replication and virion assembly. However, how NS2A participates in these two disparate processes remains unclear. In this study, we found that during ZIKV infection there are two NS2A isoforms generated, including full-length NS2A and a distinct, predominant isoform that is truncated in size, termed NS2Aα. We found that NS2Aα biogenesis was mediated by the viral NS3 protease at a unique internal cleavage motif, which is conserved across representative mosquito- and tick-borne orthoflaviviruses. Furthermore, we observed that the two NS2A isoforms play mutually exclusive roles in the viral life cycle with full-length NS2A critical for viral RNA replication and the morphology of viral replication organelles, while the NS2Aα isoform was critical for genome packaging and infectious particle production. These results provide important insights into the role of the NS2A protein and its isoforms in the ZIKV life cycle and reveal a high level of conservation across the Orthoflavivirus genus.", "abstract_shown": true, "affiliations": [ "Department of Microbiology & Immunology, University of British Columbia, Vancouver, BC, Canada.", "Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada.", "Department of Microbiology and Immunology, School of Medicine, Emory University, Atlanta, Georgia, United States of America.", "Department of Biochemistry, McGill University, Montreal, QC, Canada." ], "article_type": "Research Article", "arxiv": null, "author_total": 9, "authors": [ { "affs": [ 0, 1 ], "name": "Breanna N Landry" }, { "affs": [ 0 ], "name": "Olivia J Klann" }, { "affs": [ 0 ], "name": "Hannah C H Hauch" }, { "affs": [ 2 ], "name": "Cheyanne L Bemis" }, { "affs": [ 2 ], "name": "Jonathan E Owen" }, { "affs": [ 2 ], "name": "Jayden Harris", "orcid": "0000-0002-4682-1766" }, { "affs": [ 1 ], "name": "Trisha R Barnard" }, { "affs": [ 2 ], "name": "Christopher J Neufeldt" }, { "affs": [ 0, 1, 3 ], "name": "Selena M Sagan", "orcid": "0000-0002-8484-3632" } ], "bibtex": "https://research.draconitic.com/paper/0a1t726xhf0jj.bib", "cited_by": 0, "doi": "10.1371/journal.ppat.1014609", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014609", "id": "0a1t726xhf0jj", "issns": [ "1553-7374", "1553-7366" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014609", "language": "eng", "licence": "CC-BY", "licence_label": "CC BY", "licence_source": "article", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2739|Mosquito-borne diseases and control" ], [ "plos", "/Biology and life sciences/Biochemistry/Biosynthesis" ], [ "plos", "/Biology and life sciences/Biochemistry/Enzymology/Enzymes/Proteases" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Enzymes/Proteases" ], [ "plos", "/Biology and life sciences/Genetics/Genomics/Microbial genomics/Viral genomics" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Viral pathogens/Flaviviruses/Zika virus" ], [ "plos", "/Biology and life sciences/Microbiology/Microbial genomics/Viral genomics" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral genomics" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral replication" ], [ "plos", "/Biology and life sciences/Microbiology/Virology/Viral structure/Virions" ], [ "plos", "/Biology and life sciences/Molecular biology/Molecular biology techniques/Transfection" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/RNA viruses/Flaviviruses/Zika virus" ], [ "plos", "/Biology and life sciences/Organisms/Viruses/Viral pathogens/Flaviviruses/Zika virus" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Viral pathogens/Flaviviruses/Zika virus" ], [ "plos", "/Research and analysis methods/Database and informatics methods/Bioinformatics/Sequence analysis/Sequence motif analysis" ], [ "plos", "/Research and analysis methods/Molecular biology techniques/Transfection" ] ], "openalex": "W7221504430", "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014609&type=printable", "pmcid": "PMC13671652", "pmid": "42862866", "published": "2026-10-12", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/0a1t726xhf0jj.ris", "sources": [ { "first_seen": "2026-10-30T12:17:30Z", "native_id": "10.1371/journal.ppat.1014609", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014609" }, { "first_seen": "2026-10-30T12:19:00Z", "native_id": "MED:42862866", "source": "europepmc", "url": "https://europepmc.org/article/MED/42862866" } ], "subjects": [ { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" } ], "title": "Zika virus produces two isoforms of the NS2A protein that play mutually exclusive roles in viral replication and virion assembly", "title_text": "Zika virus produces two isoforms of the NS2A protein that play mutually exclusive roles in viral replication and virion assembly", "type": "peer-reviewed", "updated_at": "2026-10-30T12:19:00Z", "url": "https://research.draconitic.com/paper/0a1t726xhf0jj", "venue": "PLOS Pathogens", "versions": [] }