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Bemnifosbuvir (AT-527) and AT-752 are guanosine analogues currently in clinical trials against several RNA viruses. Here, we show that these drugs require a minimal set of 5 cellular enzymes for activation to their common 5′-triphosphate AT-9010, with an obligate order of reactions. AT-9010 selectively inhibits essential viral enzymes, accounting for antiviral potency. Functional and structural data at atomic resolution decipher N6-purine deamination compatible with its metabolic activation. Crystal structures of human histidine triad nucleotide binding protein 1, adenosine deaminase-like protein 1, guanylate kinase 1, and nucleoside diphosphate kinase at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with cognate precursors of AT-9010 illuminate the activation pathway from the orally available bemnifosbuvir to AT-9010, pointing to key drug–protein contacts along the activation pathway. Our work provides a framework to integrate the design of antiviral nucleotide analogues, confronting requirements and constraints associated with activation enzymes along the 5′-triphosphate assembly line. Bemnifosbuvir (AT-527) and AT-752 are broad-spectrum antiviral guanosine analogues currently in clinical trials. This study reconstitutes their intracellular 5-step enzymatic activation pathway, characterizing each enzymatic complex at atomic resolution, which provides insight into substrate binding and drug activation. [ABSTRACT FROM AUTHOR]

The large Bunyavirales order includes several families of viruses with a segmented ambisense (-) RNA genome and a cytoplasmic life cycle that starts by synthesizing viral mRNA. The initiation of transcription, which is common to all members, relies on an endonuclease activity that is responsible for cap-snatching. In La Crosse virus, an orthobunyavirus, it has previously been shown that the cap-snatching endonuclease resides in the N-terminal domain of the L protein. Orthobunyaviruses are transmitted by arthropods and cause diseases in cattle. However, California encephalitis virus, La Crosse virus and Jamestown Canyon virus are North American species that can cause encephalitis in humans. No vaccines or antiviral drugs are available. In this study, three known Influenza virus endonuclease inhibitors (DPBA, L-742,001 and baloxavir) were repurposed on the La Crosse virus endonuclease. Their inhibition was evaluated by fluorescence resonance energy transfer and their mode of binding was then assessed by differential scanning fluorimetry and microscale thermophoresis. Finally, two crystallographic structures were obtained in complex with L-742,001 and baloxavir, providing access to the structural determinants of inhibition and offering key information for the further development of Bunyavirales endonuclease inhibitors., (open access.)

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)