Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs

A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.

'Dare the boist'rous main': the role of the Belfast Newsletter in the process of emigration from Ulster to North America 1760-1800

Before the mass migrations from Ireland in the nineteenth century, earlier waves of migration in the eighteenth century saw significant numbers of people leave Ireland, predominantly from Ulster, to settle in North America. This article, using as its principal data source the Belfast News Letter ( BNL), its letters, advertisements and reports, focuses firstly on reconstructing the late eighteenth-century migration process and voyage, highlighting the barriers represented by the Atlantic Ocean. In addition to the challenges of the sea, there were problems with the ships, the ever-present danger of disease and also threats from other vessels, from privateers to press gangs. The voyage was recognized as a ‘universal dread’, and the risks taken to ‘dare the boist’rous main’ were perhaps not minimized in the pages of the BNL, whose editorial stance was antipathetic to the migration for the potential harm it caused to Ulster by removing so many of its industrious young. The second part of this article goes on to consider the newspaper’s and others’ vested interests in the emigration process, demonstrates how these were manifested in the press and sets the coverage of this very significant early emigration flow within the context of contemporary religious and colonial discourses at a period of very lively transatlantic interactions.

A non-LTE analysis of the spectra of two narrow lined main sequence stars in the SMC

An analysis of high-resolution VLT/UVES spectra of two B-type main sequence stars, NGC 346-11 and AV 304, in the Small Magellanic Cloud (SMC), has been undertaken, using the non-LTE tlusty model atmospheres to derive the stellar parameters and chemical compositions of each star. The chemical compositions of the two stars are in reasonable agreement. Moreover, our stellar analysis agrees well with earlier analyses of H II regions. The results derived here should be representative of the current base-line chemical composition of the SMC interstellar medium as derived from B-type stars.

Boron abundances in B-type stars: A test of rotational depletion during main-sequence evolution

Boron abundances have been derived for seven main-sequence B- type stars from Hubble Space Telescope STIS spectra around the B III lambda2066 line. In two stars, boron appears to be undepleted with respect to the presumed initial abundance. In one star, boron is detectable but is clearly depleted. In the other four stars, boron is undetectable, implying depletions of 1-2 dex. Three of these four stars are nitrogen enriched, but the fourth shows no enrichment of nitrogen. Only rotationally induced mixing predicts that boron depletions are unaccompanied by nitrogen enrichments. The inferred rate of boron depletion from our observations is in good agreement with these predictions. Other boron-depleted nitrogen-normal stars are identified from the literature. In addition, several boron- depleted nitrogen-rich stars are identified, and while all fall on the boron-nitrogen trend predicted by rotationally induced mixing, a majority have nitrogen enrichments that are not uniquely explained by rotation. The spectra have also been used to determine iron group (Cr, Mn, Fe, and Ni) abundances. The seven B-type stars have near-solar iron group abundances, as expected for young stars in the solar neighborhood. We have also analyzed the halo B-type star PG 0832 + 676. We find [Fe/H] = -0.88 +/- 0.10, and the absence of the B III line gives the upper limit [B/H] <-2.5. These and other published abundances are used to infer the star's evolutionary status as a post-asymptotic giant branch star.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a Ki value of 35.3 M. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor.

The coronavirus main protease, Mpro, is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compounds were performed to identify potential Mpro inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-containing peptide 2c was found to be the most potent inhibitor.

Conservation of substrate specificities among coronavirus main proteases.

The key enzyme in coronavirus replicase polyprotein processing is the coronavirus main protease, 3CL(pro). The substrate specificities of five coronavirus main proteases, including the prototypic enzymes from the coronavirus groups I, II and III, were characterized. Recombinant main proteases of human coronavirus (HCoV), transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus, avian infectious bronchitis virus and mouse hepatitis virus (MHV) were tested in peptide-based trans-cleavage assays. The determination of relative rate constants for a set of corresponding HCoV, TGEV and MHV 3CL(pro) cleavage sites revealed a conserved ranking of these sites. Furthermore, a synthetic peptide representing the N-terminal HCoV 3CL(pro) cleavage site was shown to be effectively hydrolysed by noncognate main proteases. The data show that the differential cleavage kinetics of sites within pp1a/pp1ab are a conserved feature of coronavirus main proteases and lead us to predict similar processing kinetics for the replicase polyproteins of all coronaviruses.

The main-sequence rotation-colour relation in the Coma Berenices open cluster

We present the results of a photometric survey of rotation rates in the Coma Berenices (Melotte 111) open cluster, using data obtained as part of the SuperWASP exoplanetary transit-search programme. The goal of the Coma survey was to measure precise rotation periods for main-sequence F, G and K dwarfs in this intermediate-age (~600 Myr) cluster, and to determine the extent to which magnetic braking has caused the stellar spin periods to converge. We find a tight, almost linear relationship between rotation period and J - K colour with an rms scatter of only 2 per cent. The relation is similar to that seen among F, G and K stars in the Hyades. Such strong convergence can only be explained if angular momentum is not at present being transferred from a reservoir in the deep stellar interiors to the surface layers. We conclude that the coupling time-scale for angular momentum transport from a rapidly spinning radiative core to the outer convective zone must be substantially shorter than the cluster age, and that from the age of Coma onwards stars rotate effectively as solid bodies. The existence of a tight relationship between stellar mass and rotation period at a given age supports the use of stellar rotation period as an age indicator in F, G and K stars of Hyades age and older. We demonstrate that individual stellar ages can be determined within the Coma population with an internal precision of the order of 9 per cent (rms), using a standard magnetic braking law in which rotation period increases with the square root of stellar age. We find that a slight modification to the magnetic-braking power law, P ~ t0.56, yields rotational and asteroseismological ages in good agreement for the Sun and other stars of solar age for which p-mode studies and photometric rotation periods have been published.

Rapid sympatric ecological differentiation of crater lake cichlid fishes within historic times

Background: After a volcano erupts, a lake may form in the cooled crater and become an isolated aquatic ecosystem. This makes fishes in crater lakes informative for understanding sympatric evolution and ecological diversification in barren environments. From a geological and limnological perspective, such research offers insight about the process of crater lake ecosystem establishment and speciation. In the present study we use genetic and coalescence approaches to infer the colonization history of Midas cichlid fishes (Amphilophus cf. citrinellus) that inhabit a very young crater lake in Nicaragua-the ca. 1800 year-old Lake Apoyeque. This lake holds two sympatric, endemic morphs of Midas cichlid: one with large, hypertrophied lips (~20% of the total population) and another with thin lips. Here we test the associated ecological, morphological and genetic diversification of these two morphs and their potential to represent incipient speciation.
Results: Gene coalescence analyses [11 microsatellite loci and mitochondrial DNA (mtDNA) sequences] suggest that crater lake Apoyeque was colonized in a single event from the large neighbouring great lake Managua only about 100 years ago. This founding in historic times is also reflected in the extremely low nuclear and mitochondrial genetic diversity in Apoyeque. We found that sympatric adult thin- and thick-lipped fishes occupy distinct ecological trophic niches. Diet, body shape, head width, pharyngeal jaw size and shape and stable isotope values all differ significantly between the two lip-morphs. The eco-morphological features pharyngeal jaw shape, body shape, stomach contents and stable isotopes (d15N) all show a bimodal distribution of traits, which is compatible with the expectations of an initial stage of ecological speciation under disruptive selection. Genetic differentiation between the thin- and thick-lipped population is weak at mtDNA sequence (FST = 0.018) and absent at nuclear microsatellite loci (FST < 0.001).
Conclusions: This study provides empirical evidence of eco-morphological differentiation occurring very quickly after the colonization of a new and vacant habitat. Exceptionally low levels of neutral genetic diversity and inference from coalescence indicates that the Midas cichlid population in Apoyeque is much younger (ca. 100 years or generations old) than the crater itself (ca. 1 800 years old). This suggests either that the crater remained empty for many hundreds of years after its formation or that remnant volcanic activity prevented the establishment of a stable fish population during the early life of the crater lake. Based on our findings of eco-morphological variation in the Apoyeque Midas cichlids, and known patterns of adaptation in Midas cichlids in general, we suggest that this population may be in a very early stage of speciation (incipient species), promoted by disruptive selection and ecological diversification.

Characterization of bafinivirus main protease autoprocessing activities

The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (Mpro) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV Mpro is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-ß-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV Mpro autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln?(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of Mpro, resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative Mpro autoprocessing sites has not been described previously for other nidovirus Mpro domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales.

MML 53: a new low-mass, pre-main sequence eclipsing binary in the Upper Centaurus-Lupus region discovered by SuperWASP

We announce the discovery of a new low-mass, pre-main sequence eclipsing binary, MML 53. Previous observations of MML 53 found it to be a pre-main sequence spectroscopic multiple associated with the 15-22 Myr Upper Centaurus-Lupus cluster. We identify the object as an eclipsing binary for the first time through the analysis of multiple seasons of time series photometry from the SuperWASP transiting planet survey. Re-analysis of a single archive spectrum shows MML 53 to be a spatially unresolved triple system of young stars which all exhibit significant lithium absorption. Two of the components comprise an eclipsing binary with period, P = 2.097891(6) ± 0.000005 and mass ratio, q ~ 0.8. Here, we present the analysis of the discovery data.