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Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3′-5′ exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3′-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5′-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

The pH-sensitive mechanism that gets alphaviruses into host cells Alphaviruses are significant animal and human pathogens — as demonstrated in recent outbreaks of infection with the mosquito-borne Chikungunya virus in India and southeast Asia. The E1 and E2 glycoproteins of alphaviruses are central to the way the virus infects host cells. The E1/E2 heterodimers that form spikes on the virus surface dissociate in the acidic conditions found in the internal vesicles of host cells, and E1 triggers infection by fusing with the endosomal membrane. Félix Rey and colleagues present the structure of Chikungunya virus envelope glycoprotein at neutral pH, and Michael Rossmann and colleagues reveal the structure of the envelope proteins of Sindbis virus at low pH. Sindbis virus can cause fever in humans and is the most extensively studied alphavirus. Comparison of the two structures provides insight into how fusion activation is controlled and points to possible vaccine targets. The E1 and E2 glycoproteins of alphaviruses form heterodimers and assemble into spikes on the virus surface, which mediate receptor binding and endocytosis. When the virion encounters acidic pH in the endosome E1 and E2 dissociate and E1 triggers fusion with the endosomal membrane. Two papers now provide the first crystal structures for glycoprotein complexes incorporating E2 at acidic and neutral pH, respectively. Together they provide insight into how fusion activation is controlled in alphaviruses. Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused widespread outbreaks of debilitating human disease in the past five years 1 . CHIKV invasion of susceptible cells is mediated by two viral glycoproteins, E1 and E2, which carry the main antigenic determinants and form an icosahedral shell at the virion surface. Glycoprotein E2, derived from furin cleavage of the p62 precursor into E3 and E2, is responsible for receptor binding, and E1 for membrane fusion. In the context of a concerted multidisciplinary effort to understand the biology of CHIKV 2 , here we report the crystal structures of the precursor p62–E1 heterodimer and of the mature E3–E2–E1 glycoprotein complexes. The resulting atomic models allow the synthesis of a wealth of genetic, biochemical, immunological and electron microscopy data accumulated over the years on alphaviruses in general. This combination yields a detailed picture of the functional architecture of the 25 MDa alphavirus surface glycoprotein shell. Together with the accompanying report on the structure of the Sindbis virus E2–E1 heterodimer at acidic pH (ref. 3 ), this work also provides new insight into the acid-triggered conformational change on the virus particle and its inbuilt inhibition mechanism in the immature complex.

The X-ray crystal structure of influenza C virus polymerase, captured in a closed, pre-activation confirmation, is solved at 3.9 Å resolution; comparison with previous RNA-bound structures reveals large conformational changes associated with RNA binding and activation, and illustrates the notable flexibility of the influenza virus RNA polymerase. Atomic structure of influenza C virus RNA polymerase Erwin Fodor and colleagues report the crystal structure of influenza C virus RNA polymerase, captured in a closed, pre-activation conformation. This closed conformation appears very different from previously reported crystal structures of influenza A and B virus polymerases, which contained the RNA promoter. Influenza C infects pigs and humans, but is rarer than the A and B viruses and causes a less severe form of flu. Comparison of the new structure with the previous RNA-bound structures identifies large conformational changes associated with RNA binding and activation, illustrating the flexibility of the influenza virus RNA polymerase. Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome 1 . In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching 2 . Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5′ and 3′ termini of viral genome segments), showing FluPol in transcription pre-initiation states 3 , 4 . Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new ‘closed’ conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols 3 , 4 . The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca²⁺ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca²⁺ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.

The respiratory syncytial virus (RSV) is an important human pathogen, yet neither a vaccine nor effective therapies are available to treat infection. To help elucidate the replication mechanism of this RNA virus, we determined the three-dimensional (3D) crystal structure at 3.3 Å resolution of a decameric, annular ribonucleoprotein complex of the RSV nucleoprotein (N) bound to RNA. This complex mimics one turn of the viral helical nucleocapsid complex, which serves as template for viral RNA synthesis. The RNA wraps around the protein ring, with seven nucleotides contacting each N subunit, alternating rows of four and three stacked bases that are exposed and buried within a protein groove, respectively. Combined with electron microscopy data, this structure provides a detailed model for the RSV nucleocapsid, in which the bases are accessible for readout by the viral polymerase. Furthermore, the nucleoprotein structure highlights possible key sites for drug targeting.

Arenaviruses are important agents of zoonotic disease worldwide. The virions expose a tripartite envelope glycoprotein complex at their surface, formed by the glycoprotein subunits GP1, GP2 and the stable signal peptide. This complex is responsible for binding to target cells and for the subsequent fusion of viral and host-cell membranes for entry. During this process, the acidic environment of the endosome triggers a fusogenic conformational change in the transmembrane GP2 subunit of the complex. We report here the crystal structure of the recombinant GP2 ectodomain of the lymphocytic choriomeningitis virus, the arenavirus type species, at 1.8-Å resolution. The structure shows the characteristic trimeric coiled coil present in class I viral fusion proteins, with a central stutter that allows a close structural alignment with most of the available structures of class I and III viral fusion proteins. The structure further shows a number of intrachain salt bridges stabilizing the postfusion hairpin conformation, one of which involves an aspartic acid that appears released from a critical interaction with the stable signal peptide upon low pH activation.

Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a 'cap-snatching' mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.

The ability to utilize a hybrid‐photon‐counting detector to its full potential can significantly influence data quality, data collection speed, as well as development of elaborate data acquisition schemes. This paper facilitates the optimal use of EIGER2 detectors by providing theory and practical advice on (i) the relation between detector design, technical specifications and operating modes, (ii) the use of corrections and calibrations, and (iii) new acquisition features: a double‐gating mode, 8‐bit readout mode for increasing temporal resolution, and lines region‐of‐interest readout mode for frame rates up to 98 kHz. Examples of the implementation and application of EIGER2 at several synchrotron sources (ESRF, PETRA III/DESY, ELETTRA, AS/ANSTO) are presented: high accuracy of high‐throughput data in serial crystallography using hard X‐rays; suppressing higher harmonics of undulator radiation, improving peak shapes, increasing data collection speed in powder X‐ray diffraction; faster ptychography scans; and cleaner and faster pump‐and‐probe experiments. A presentation of how EIGER2 detectors work and how to best use their newly released features for fast image acquisitions and advanced acquisition schemes.

Neurofibromatosis type 2 (NF2) is a tumor-forming disease of the nervous system caused by deletion or by loss-of-function mutations in NF2 , encoding the tumor suppressing protein neurofibromin 2 (also known as schwannomin or merlin). Neurofibromin 2 is a member of the ezrin, radixin, moesin (ERM) family of proteins regulating the cytoskeleton and cell signaling. The correlation of the tumor-suppressive function and conformation (open or closed) of neurofibromin 2 has been subject to much speculation, often based on extrapolation from other ERM proteins, and controversy. Here we show that lipid binding results in the open conformation of neurofibromin 2 and that lipid binding is necessary for inhibiting cell proliferation. Collectively, our results provide a mechanism in which the open conformation is unambiguously correlated with lipid binding and localization to the membrane, which are critical for the tumor-suppressive function of neurofibromin 2, thus finally reconciling the long-standing conformation and function debate. Neurofibromin 2 (NF2) is a tumour suppressor that inhibits cell growth. Here the authors combine functional, biochemical, and structural studies and show that lipid-bound NF2 adopts an open conformation and that NF2 lipid binding is required for inhibition of cell proliferation.