Highlights d Structures of SARS-CoV-2 RNA polymerase in complexes with RNA revealed d Conformational changes in nsp8 and its interaction with the exiting RNA are observed d Incorporation and delayed-chain-termination mechanism of remdesivir is elucidated d Transition model from primase complex to polymerase complex is proposed

Positive-strand RNA viruses include a large number of human and animal pathogens whose essential RNA-dependent RNA polymerases (RdRPs) share a structurally homologous core with an encircled active site. RdRPs are targets for antiviral drug development, but these efforts are hindered by limited structural information about the RdRP catalytic cycle. To further our understanding of RdRP function, we assembled, purified, and then crystallized poliovirus elongation complexes after multiple rounds of nucleotide incorporation. Here we present structures capturing the active polymerase and its nucleotide triphosphate complexes in four distinct states, leading us to propose a six-state catalytic cycle involving residues that are highly conserved among positive-strand RNA virus RdRPs. The structures indicate that RdRPs use a fully prepositioned templating base for nucleotide recognition and close their active sites for catalysis using a novel structural rearrangement in the palm domain. The data also suggest that translocation by RDRPs may not be directly linked to the conformational changes responsible for active site closure and reopening.replication | picornavirus P ositive-strand RNA viruses cause diseases such as the common cold, acute hepatitis A, chronic hepatitis C, hemorrhagic fevers, meningitis, encephalitis, and paralytic poliomyelitis. The genome replication cycle of these viruses is entirely RNA-based, with replication being carried out by virally encoded RNA-dependent RNA polymerases that retain several hallmark polymerase sequence motifs within a ≈500-residue core structure composed of the typical polymerase palm, fingers, and thumb domains (1-5). A unique and conserved aspect of RNA-dependent RNA polymerase (RdRP) structures is that the fingers domain reaches across the palm to interact with the top of the thumb, encircling the active site.Extensive biochemical and structural studies of polymerases in general have defined the catalytic cycle as a multistep process composed of initial NTP binding to an "open" conformation of the enzyme-template complex, a recognition event to ensure that only the correct nucleotide triggers the formation of the catalytically competent "closed" conformation, catalysis, and then an opening of the active site that is usually accompanied by translocation of the nucleic acid (6-8). RdRPs also use this multistep catalytic process, but the encircled active site structure makes it unlikely that major swinging motions of the fingers domain act to reposition the nascent base pair from an initial preinsertion site into the catalytic site, as is seen in other classes of single-subunit polymerases (9-12). Biochemical studies of poliovirus and foot-and-mouth disease virus (FMDV) RdRPs have shown that addition of the first one or two nucleotides onto a primer is the key initiation step on the pathway to forming a very stable and highly processive elongation complex (13,14).Despite the vast number of viral RdRP structures that have been solved over the past fifteen years, little is known a...

The flavivirus NS5 harbors a methyltransferase (MTase) in its N-terminal ≈265 residues and an RNA-dependent RNA polymerase (RdRP) within the C-terminal part. One of the major interests and challenges in NS5 is to understand the interplay between RdRP and MTase as a unique natural fusion protein in viral genome replication and cap formation. Here, we report the first crystal structure of the full-length flavivirus NS5 from Japanese encephalitis virus. The structure completes the vision for polymerase motifs F and G, and depicts defined intra-molecular interactions between RdRP and MTase. Key hydrophobic residues in the RdRP-MTase interface are highly conserved in flaviviruses, indicating the biological relevance of the observed conformation. Our work paves the way for further dissection of the inter-regulations of the essential enzymatic activities of NS5 and exploration of possible other conformations of NS5 under different circumstances.

Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTPinduced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the templateproduct duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases.RNA-dependent RNA polymerase | nucleotide addition cycle | translocation intermediate | enterovirus 71 | crystal structure I n recent years, several notable emerging infectious diseases have been caused by RNA viruses, including highly pathogenic avian influenza viruses, Ebola virus, and Middle East respiratory syndrome coronavirus. RNA viruses are quite diverse in virus particle and genome structure and in virus entry and assembly mechanisms. However, they do share fundamental features in their genome replication and transcription, using a virally encoded RNAdependent RNA polymerase (RdRP) to carry out the biosynthesis of an RNA product directed by an RNA template. Although the genome replication machinery often requires the participation of other factors, typically at the initiation phase of synthesis, the RdRP governs the elongation phase of synthesis that includes thousands of efficient nucleotide addition cycles (NACs). Viral RdRPs vary greatly in size and structural organization, from the ∼50-kDa picornavirus 3D pol (1, 2), to the ∼100-kDa flavivirus NS5 that contains a naturally fused methyltransferase domain (3), to the ∼250-kDa nonsegmented negative-strand RNA virus L protein harboring at least three enzyme modules (4) and the ∼260-kDa three-subunit PA-PB1-PB2 influenza virus replicase complex (5). On the other hand, all RdRPs share a 50-to 70-kDa polymerase core that forms a unique encircled right-hand structure with palm, fingers, and thumb domains. Among the seven classic RdRP catalytic motifs, A-E are within the most conserved palm domain, and F and G are located in the fingers; they are all arranged similarly around the active site (6-9). The structural conservation of the RdRP polymerase core and the seven motifs form the basis for understanding the common features in viral RdRP catalytic mechanism and for finding intervention strategies targeting these enzymes with possible broad-spectrum potential.As with other classes of nucleic acid polymerases, the viral RdRP elongation NAC comprises sequential steps of in...