Explore the vast range of titles available.

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp8 2-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3′ end of the RNA product strand. Its modified ribose group (2′-fluoro, 2′-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

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 broad spectrum antiviral potency. Functional and structural data at atomic resolution decipher N6-purine deamination compatible with metabolic activation by human ADALP1. Crystal structures of human HINT1, ADALP1, GUK1, and NDPK at 2.09, 2.44, 1.76, and 1.9 A 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.

Large dsDNA viruses from the Naldaviricetes class are currently composed of four viral families infecting insects and/or crustaceans. Since the 1970s, particles described as filamentous viruses (FVs) have been observed by electronic microscopy in several species of Hymenoptera parasitoids but until recently, no genomic data was available. This study provides the first comparative morphological and genomic analysis of these FVs. We analyzed the genomes of seven FVs, six of which were newly obtained, to gain a better understanding of their evolutionary history. We show that these FVs share all genomic features of the Naldaviricetes while encoding five specific core genes that distinguish them from their closest relatives, the Hytrosaviruses. By mining public databases, we show that FVs preferentially infect Hymenoptera with parasitoid lifestyle and that these viruses have been repeatedly integrated into the genome of many insects, particularly Hymenoptera parasitoids, overall suggesting a long-standing specialization of these viruses to parasitic wasps. Finally, we propose a taxonomical revision of the class Naldaviricetes in which FVs related to the Leptopilina boulardi FV constitute a fifth family. We propose to name this new family, Filamentoviridae.

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.

Arenaviruses are emerging enveloped negative-sense RNA viruses which cause neurological and hemorrhagic diseases in humans. Currently there is no FDA approved vaccine or therapeutic agent except ribavirin which must be administered early during infection for optimum efficacy. A hallmark for arenavirus infection is a rapid and efficient immune-suppression mediated by the exonuclease domain encoded by the nucleoprotein. This exonuclease is therefore an attractive target for the design of novel antiviral drugs since exonuclease inhibitors might not only have a direct effect on the enzyme but could also boost viral clearance through stimulation of the innate immune system of the host cell. Here we have used an in silico screening and an enzymatic assay to identify a novel, specific but weak inhibitor of the arenavirus exonuclease with IC 50 values of 65.9 µM and 68.6 µM for Mopeia virus and Lymphocytic choriomeningitis virus, respectively. We further characterized this finding with crystallographic and docking approaches. This study serves as a proof of concept and may have assigned a new therapeutic purpose for the bisphosphonate family and therefore paves the way for the development of inhibitors against Arenaviridae.

Influenza A viruses (IAVs) use diverse mechanisms to interfere with cellular gene expression. Although many RNA-seq studies have documented IAV-induced changes in host mRNA abundance, few were designed to allow an accurate quantification of changes in host mRNA splicing. Here, we show that IAV infection of human lung cells induces widespread alterations of cellular splicing, with an overall increase in exon inclusion and decrease in intron retention. Over half of the mRNAs that show differential splicing undergo no significant changes in abundance or in their 3 end termination site, suggesting that IAVs can specifically manipulate cellular splicing. Among a randomly selected subset of 21 IAV-sensitive alternative splicing events, most are specific to IAV infection as they are not observed upon infection with VSV, induction of interferon expression or induction of an osmotic stress. Finally, the analysis of splicing changes in RED-depleted cells reveals a limited but significant overlap with the splicing changes in IAV-infected cells. This observation suggests that hijacking of RED by IAVs to promote splicing of the abundant viral NS1 mRNAs could partially divert RED from its target mRNAs. All our RNA-seq datasets and analyses are made accessible for browsing through a user-friendly Shiny interface (http://virhostnet.prabi. fr:3838/shinyapps/flu-splicing or https://github.com/ cbenoitp/flu-splicing).

Summary How viruses from the Coronaviridae family initiate viral RNA synthesis is unknown. Here we show that the SARS-CoV-1 and −2 Nidovirus RdRp-Associated Nucleotidyltransferase (NiRAN) domain on nsp12 uridylates the viral cofactor nsp8, forming a UMP-Nsp8 covalent intermediate that subsequently primes RNA synthesis from a poly(A) template; a protein-priming mechanism reminiscent of Picornaviridae enzymes. In parallel, the RdRp active site of nsp12 synthesizes a pppGpU primer, which primes (-)ssRNA synthesis at the precise genome-poly(A) junction. The guanosine analogue 5’-triphosphate AT-9010 (prodrug: AT-527) tightly binds to the NiRAN and inhibits both nsp8-labeling and the initiation of RNA synthesis. A 2.98 Å resolution Cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-(nsp8)2 /RNA/NTP quaternary complex shows AT-9010 simultaneously binds to both NiRAN and RdRp active site of nsp12, blocking their respective activities. AT-527 is currently in phase II clinical trials, and is a potent inhibitor of SARS-CoV-1 and −2, representing a promising drug for COVID-19 treatment. Competing Interest Statement S.G., A.M., K.L. and J.P.S. are employees of ATEA Pharmaceuticals, Inc. The other authors declare no competing interests.