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While alphavirus-based saRNA systems are widely used, they suffer from limitations, including large genome size, complex replicase machinery, and cellular toxicity. Nodamura virus (NoV) offers a promising alternative due to its compact genome (3.2 kb) and low cytotoxicity. This study aimed to elucidate NoV RNA1 replication mechanisms and develop a novel NoV-based saRNA vector platform. Methods: We established a Schizosaccharomyces pombe system to investigate NoV RNA1 replication and protein A localization. N-terminal deletion mutants and ER-targeting chimeras were constructed to characterize membrane targeting determinants. Based on mechanistic insights, we developed NovaVec by inserting transgenes at the RNA3422 site within the subgenomic RNA3 region. In vivo performance was evaluated using lipid nanoparticle-encapsulated NovaVec expressing nanoluciferase or monkeypox A33R antigen in BALB\/c mice. Results: We identified redundant mitochondrial targeting domains (amino acids 2-15 and 16-33) in NoV protein A, where either domain was sufficient for proper localization and replication. The replication machinery could be functionally redirected to the endoplasmic reticulum while maintaining replication competence. Lipid nanoparticle-encapsulated NovaVec achieved sustained transgene expression for 54 days in mice, significantly outperforming conventional mRNA vectors that lost signal within 14 days. The NovaVec-based monkeypox A33R vaccine elicited robust antigen-specific humoral immunity with titers reaching approximately 1:12,800 following booster immunization. Conclusions: With its compact genome encoding only a single replicase protein, minimal cytopathic effects, and demonstrated capacity for long-term protein expression, NovaVec represents a highly promising next-generation saRNA platform for vaccines.<\/jats:p>","DOI":"10.3390\/vaccines14060532","type":"journal-article","created":{"date-parts":[[2026,6,16]],"date-time":"2026-06-16T00:49:01Z","timestamp":1781570941000},"page":"532","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Deciphering the Nodamura virus Protein A Function in Schizosaccharomyces pombe and Engineering a Novel Self-Amplifying RNA (saRNA) Vector NovaVec for Vaccine Development"],"prefix":"10.3390","volume":"14","author":[{"given":"Xueyao","family":"Song","sequence":"first","affiliation":[{"name":"Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruihan","family":"Liu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuo","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuying","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Neurology, Fujian Institute of Neurology, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanting","family":"Qu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Niubing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuan","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Synthetic Biology, State Key Laboratory of Plant Trait Design, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China"},{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangping","family":"Yao","sequence":"additional","affiliation":[{"name":"Department of Neurology, Fujian Institute of Neurology, The First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1498-7059","authenticated-orcid":false,"given":"Pei","family":"Hao","sequence":"additional","affiliation":[{"name":"University of Chinese Academy of Sciences, Beijing 100039, China"},{"name":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/j.stem.2013.06.001","article-title":"Efficient generation of human iPSCs by a synthetic self-replicative RNA","volume":"13","author":"Yoshioka","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"7641767","DOI":"10.1155\/2019\/7641767","article-title":"Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA","volume":"2019","author":"Steinle","year":"2019","journal-title":"Stem Cells Int."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/j.ymthe.2017.11.017","article-title":"Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses","volume":"26","author":"Vogel","year":"2018","journal-title":"Mol. 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