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<p>The hantavirus L protein is a viral polymerase essential for viral transcription and replication; however, its expression in mammalian cells has been notoriously difficult. 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 <i>cis-</i>, and <i>trans-</i>regulatory effects in functional analyses, such as in minigenome assays, to prevent data misinterpretation.</p>

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Hantavirus L protein exhibits shutoff activity mediated by its N-terminal endonuclease domain

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月14日

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  • Kohei Oishi,
  • Tomoki Yoshikawa,
  • Satoshi Taniguchi,
  • Madoka Kawahara,
  • Takeshi Kurosu,
  • Masayuki Shimojima

摘要

The hantavirus L protein is a viral polymerase essential for viral transcription and replication; however, its expression in mammalian cells has been notoriously difficult. 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 cis-, and trans-regulatory effects in functional analyses, such as in minigenome assays, to prevent data misinterpretation.