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In this study, we achieved robust plasmid-based expression of the L protein by combining a T7-driven system with mutations that reduce endonuclease activity. This strategy was pivotal, as conventional RNA polymerase II (Pol II)-dependent systems failed to yield detectable expression. Although wild-type L protein was barely detectable, its presence suppressed co-expressed genes, suggesting a potent host shutoff activity that inhibits both trans-gene and its own (cis-) expression. Leveraging functional homology to the influenza PA-X protein, we identify amino acid residues essential for the shutoff activity of L protein by using its N-terminal fragment, which can be expressed via standard Pol II-dependent systems. Our mutagenesis analysis established a toolset for the predictable fine-tuning of shutoff activity and L protein expression levels, facilitating a detailed analysis of the interplay between polymerase activity and viral replication. These findings elucidate the mechanisms underlying the difficulty in expressing the hantavirus L protein and emphasize the necessity of accounting for these\n                    <jats:italic>cis-<\/jats:italic>\n                    , and\n                    <jats:italic>trans-<\/jats:italic>\n                    regulatory effects in functional analyses, such as in minigenome assays, to prevent data misinterpretation.\n                  <\/jats:p>","DOI":"10.1038\/s41598-026-47692-3","type":"journal-article","created":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T12:25:12Z","timestamp":1776169512000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Hantavirus L protein exhibits shutoff activity mediated by its N-terminal endonuclease domain"],"prefix":"10.1038","volume":"16","author":[{"given":"Kohei","family":"Oishi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomoki","family":"Yoshikawa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Satoshi","family":"Taniguchi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Madoka","family":"Kawahara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeshi","family":"Kurosu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masayuki","family":"Shimojima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"47692_CR1","doi-asserted-by":"publisher","first-page":"2559","DOI":"10.3390\/v2122559","volume":"2","author":"B Hjelle","year":"2010","unstructured":"Hjelle, B. & Torres-P\u00e9rez, F. 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