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Summary Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors., Graphical Abstract, Highlights • Cryo-EM structure of RSV L bound by tetrameric RSV P solved to 3.2 Å • P tetramer adopts an asymmetric tentacular arrangement when bound to L • L priming loop adopts elongation-compatible state without PRNTase-RdRp separation • Structure rationalizes escape from small-molecule antivirals, Respiratory syncytial virus (RSV) remains a leading cause of bronchiolitis and hospitalization, especially of infants. Gilman et al. present a 3.2-Å cryo-EM structure of the RSV L polymerase in complex with the P phosphoprotein—components of the core viral replication machinery that represent an attractive target for the development of therapeutic agents.

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5'-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5'-triphosphate species. ALS-8112 is a 2'- and 4'-modified nucleoside analog, prompting us to investigate dCK recognition of other 2'- and 4'-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure-activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery.

Paramyxoviruses represent a family of RNA viruses causing significant human diseases. These include measles virus, the most infectious virus ever reported, in addition to parainfluenza virus, and other emerging viruses. Paramyxoviruses likely share common replication machinery but their mechanisms of RNA biosynthesis activities and details of their complex polymerase structures are unknown. Mechanistic and functional details of a paramyxovirus polymerase would have sweeping implications for understanding RNA virus replication and for the development of new antiviral medicines. To study paramyxovirus polymerase structure and function, we expressed an active recombinant Nipah virus (NiV) polymerase complex assembled from the multifunctional NiV L protein bound to its phosphoprotein cofactor. NiV is an emerging highly pathogenic virus that causes severe encephalitis and has been declared a global public health concern due to its high mortality rate. Using negative-stain electron microscopy, we demonstrated NiV polymerase forms ring-like particles resembling related RNA polymerases. We identified conserved sequence elements driving recognition of the 3′-terminal genomic promoter by NiV polymerase, and leading to initiation of RNA synthesis, primer extension, and transition to elongation mode. Polyadenylation resulting from NiV polymerase stuttering provides a mechanistic basis for transcription termination. It also suggests a divergent adaptation in promoter recognition between pneumo- and paramyxoviruses. The lack of available antiviral therapy for NiV prompted us to identify the triphosphate forms of R1479 and GS-5734, two clinically relevant nucleotide analogs, as substrates and inhibitors of NiV polymerase activity by delayed chain termination. Overall, these findings provide low-resolution structural details and the mechanism of an RNA polymerase from a previously uncharacterized virus family. This work illustrates important functional differences yet remarkable similarities between the polymerases of nonsegmented negative-strand RNA viruses., Author summary RNA viruses replicate and transcribe their genomes using complex enzymatic machines known as RNA-dependent RNA polymerases. The chemical reactions driving nucleotide addition are shared among nucleic acid polymerases but the underlying mechanisms of RNA biosynthesis and the complex polymerase structures are diverse. Of these RNA viruses is the paramyxovirus family, which includes major human pathogens. Paramyxoviruses have common biological and genetic properties but little is known about their replication machinery. Insights into the structure, function, and mechanisms of RNA synthesis of one paramyxovirus polymerase will likely extend to the entire virus family. An emerging, highly pathogenic paramyxovirus is Nipah virus (NiV), which causes encephalitis in humans. We have purified NiV polymerase, probed its enzymatic and biophysical properties and developed it as a model paramyxovirus polymerase. We investigated template strand sequence elements driving RNA biosynthesis for NiV polymerase and obtained a snapshot of NiV polymerase molecular organization using electron microscopy to provide the first structural information on a paramyxovirus polymerase. This work extends previous knowledge by producing the first recombinant paramyxovirus polymerase and using this protein in enzymatic assays to highlight key functional and structural characteristics for the design of new medicines.

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