Deciphering the Nodamura virus Protein A Function in Schizosaccharomyces pombe and Engineering a Novel Self-Amplifying RNA (saRNA) Vector NovaVec for Vaccine Development

作者：Xueyao Song, Ruihan Liu, Z ZHANG, Yuying Pan, Wanting Qu, Niubing Zhang, Xuan Li, Xiangping Yao, Pei Hao · 发表于：Vaccines · 年份：2026 · DOI：10.3390/vaccines14060532 · 研究领域：Poxvirus research and outbreaks、Plant Virus Research Studies、RNA Interference and Gene Delivery

Background/Objectives: Self-amplifying RNA (saRNA) vectors enable high-level transgene expression from minimal initial doses. 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 mRN...