Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation

Water vapour in the atmosphere of a transiting extrasolar planet

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches

New statistical potential for quality assessment of protein models and a survey of energy functions

Abstract Background Scoring functions, such as molecular mechanic forcefields and statistical potentials are fundamentally important tools in protein structure modeling and quality assessment. Results The performances of a number of publicly available scoring functions are compared with a statistical rigor, with an emphasis on knowledge-based potentials. We explored the effect on accuracy of alternative choices for representing interaction center types and other features of scoring functions, such as using information on solvent accessibility, on torsion angles, accounting for secondary structure preferences and side chain orientation. Partially based on the observations made, we present a novel residue based statistical potential, which employs a shuffled reference state definition and takes into account the mutual orientation of residue side chains. Atom- and residue-level statistical potentials and Linux executables to calculate the energy of a given protein proposed in this work can be downloaded from http://www.fiserlab.org/potentials. Conclusions Among the most influential terms we observed a critical role of a proper reference state definition and the benefits of including information about the microenvironment of interaction centers. Molecular mechanical potentials were also tested and found to be over-sensitive to small local imperfections in a structure, requiring unfeasible long energy relaxation before energy scores started to correlate with model quality.</p

Spontaneous Cutaneous Rupture of a Pyogenic Liver Abscess

Spontaneous rupture of a pyogenic liver abscess is a rare entity reported. Spontaneous cutaneous rupture of a pyogenic liver abscess is even a rarer clinical entity not well documented in the literature. A patient having spontaneous cutaneous rupture of a pyogenic liver abscess is reported. The condition is rare and serious because of the possible delay in diagnosis. Recently, with the advent of ultrasound (USG) and computed tomography (CT) scan, there has been improvement in the rate of early diagnosis of liver abscess as well as in prognosis. The clinical presentation is variable; in some cases there are delays, misdiagnoses, or incidental recovery. Per cutaneous catheter drainage under USG or CT guidance has become the standard of care

Crystal structure of isopenicillin <i>N</i> synthase is the first from a new structural family of enzymes

Penicillin antibiotics are all produced from fermentation-derived penicillins because their chemical synthesis is not commercially viable. The key step in penicillin biosynthesis, in which both the beta-lactam and thiazolidine rings of the nucleus are created, is mediated by isopenicillin N synthase (IPNS), which binds ferrous iron and uses dioxygen as a cosubstrate. In a unique enzymatic step, with no chemical precedent, IPNS catalyses the transfer of four hydrogen atoms from its tripeptide substrate to dioxygen forming, in a single reaction, the complete bicyclic nucleus of the penicillins. We now report the structure of IPNS complexed with manganese, which reveals the active site is unusually buried within a 'jelly-roll' motif and lined by hydrophobic residues, and suggest how this structure permits the process of penicillin formation. Sequence analyses indicate IPNS, 1-aminocyclopropane-1-carboxylic acid oxidase and many of the 2-oxo-acid-dependent oxygenases contain a conserved jelly-roll motif, forming a new structural family of enzymes

Structure at 2.8 Â resolution of F1-ATPase from bovine heart mitochondria

In the crystal structure of bovine mitochondrial F-1-ATPase determined at 2.8 Angstrom resolution, the three catalytic beta-subunits differ in conformation and in the bound nucleotide. The structure supports a catalytic mechanism in intact ATP synthase in which the three catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of the alpha(3) beta(3) subassembly relative to an alpha-helical domain of the gamma-subunit