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 Å2 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.

Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This ∼1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca 2þ , a cation mandatory for infection, the RBPs rotated 200°downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.crystal structure | electon microscopy | Lactococcus lactis | Siphoviridae | bacteriophage

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 Ca 2þ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca 2þ 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.bacteriophage | crystal structure | Lactococcus lactis | siphoviridae | viral infection B acterial viruses (phages) are elegant nanomachines infecting their hosts with high specificity and efficiency. The vast majority of them belong to the Caudovirales order and possess a tail appendage used to recognize the host and ensure genome delivery. This organelle seems to be responsible for the unequaled efficacy of these virions, as compared to eukaryotic viruses, as each phage particle can infect a target cell (1). The initial events of the infection process have been characterized for a limited number of phages demonstrating that dramatic conformational changes at the distal tail end accompany the DNA injection event (2-7). Binding of myophage T4 to the host lipopolysaccharides induces dramatic rearrangements of its tail extremity allowing irreversible commitment to the target cell followed by puncturing of its surface (3, 5). The emerging picture for Podoviridae is that after attachment to the cell envelope, the minor phage proteins are ejected to form a tube across the periplasm thereby directing the viral DNA into the host cytoplasm (8). Siphophage p2, belonging to the 936-species of lactococcal phages, exhibits a Ca 2þ -mediated activation mechanism inducing a 200°rotation of the receptor-binding proteins (RBPs) to establish multiple interactions with host saccharides and initiate infection (7). Here we report the crystal structure of the baseplate from the lactoccocal phage TP901-1 (P335-species) at 3.8 Å resolution and show that it is in a conformation ready for host adhesion, ruling out the involvement of a Ca 2þ -mediated or any other major conformational changes of this organelle at this initial step. We extended these conclusions by demonstrating that authentic virions can infect their host with maximal efficacy in the absence of added Ca 2þ and that this behavior is conserved among the TP901-1 phage species (P335). We therefore suggest that TP901-1-like phages do not rely on baseplate conformational changes for host adsorption, in contrast to what has been observed for ...