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

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.