Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. One protein known to play a critical role in the coronavirus life cycle is nonstructural protein 1 (nsp1).

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae. We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward. Significance It is well known that ribosomal RNA processing is directly impacted by the rate of transcription elongation by RNA polymerase I (Pol I). To understand how these processes are orchestrated, we must carefully define transcription elongation properties in vitro and in living cells. Here, we characterize DNA sequence elements that pause and terminate Pol I transcription in vitro. We also establish methods for analyzing Pol I transcription elongation properties in vivo using native elongating transcript sequencing (NETSeq). Our NETSeq data revealed frequent pausing by Pol I and decreased Pol I occupancy at G residues, suggesting unequal rates of nucleotide incorporation by the enzyme. These findings redefine our understanding of Pol I transcription elongation and its heterogeneity in vivo.

The influenza virus is a significant public health concern causing 250,000–500,000 deaths worldwide each year. Its ability to change quickly results in the potential for rapid generation of pandemic strains for which most individuals would have no antibody protection. This pandemic potential highlights the need for the continuous development of new drugs against influenza virus. As an essential component, and well-established virulence determinant, the non-structural protein 1 (NS1) of influenza virus is a highly prioritized target for the development of anti-influenza compounds. Here, we used NMR to determine that the NS1 effector domain (NS1^ED) derived from the A/Brevig Mission/1/1918 (H1N1) strain of influenza (1918^H1N1) binds to two previously described anti-influenza compounds A9 (JJ3297) and A22. We then used X-ray crystallography to determine the three-dimensional structure of the 1918^H1N1 NS1^ED. Furthermore, we mapped the A9/A22-binding site onto our 1918^H1N1 NS1^ED structure and determined that A9 and A22 interact with the NS1^ED in the hydrophobic pocket known to facilitate binding to the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), suggesting that the two compounds likely attenuate influenza replication by inhibiting the NS1^ED–CPSF30 interaction. Finally, our structure revealed that NS1^ED could dimerize via an interface that we termed the α₃-α₃ dimer. Taken together, the findings presented here provide strong evidence for the mechanism of action of two anti-influenza compounds that target NS1 and contribute significant structural insights into NS1 that we hope will promote and inform the development and optimization of influenza therapies based on A9/A22.