A simple reverse genetics method to generate recombinant coronaviruses

Engineering recombinant viruses is a pre‐eminent tool for deciphering the biology of emerging viral pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, the large size of coronavirus genomes renders the current reverse genetics methods challenging. Here, we describe a simple method based on “infectious subgenomic amplicons” (ISA) technology to generate recombinant infectious coronaviruses with no need for reconstruction of the complete genomic cDNA and apply this method to SARS‐CoV‐2 and also to the feline enteric coronavirus. In both cases we rescue wild‐type viruses with biological characteristics similar to original strains. Specific mutations and fluorescent red reporter genes can be readily incorporated into the SARS‐CoV‐2 genome enabling the generation of a genomic variants and fluorescent reporter strains for in vivo experiments, serological diagnosis, and antiviral assays. The swiftness and simplicity of the ISA method has the potential to facilitate the advance of coronavirus reverse genetics studies, to explore the molecular biological properties of the SARS‐CoV‐2 variants, and to accelerate the development of effective therapeutic reagents.     

An analysis of determinants of amino acids substitution rates in bacterial proteins

The variation of amino acid substitution rates in proteins depends on several variables. Among these, the protein's expression level, functional category, essentiality, or metabolic costs of its amino acid residues may play an important role. However, the relative importance of each variable has not yet been evaluated in comparative analyses. To this aim, we made regression analyses combining data available on these variables and on evolutionary rates, in two well-documented model bacteria, Escherichia coli and Bacillus subtilis. In both bacteria, the level of expression of the protein in the cell was by far the most important driving force constraining the amino acids substitution rate. Subsequent inclusion in the analysis of the other variables added little further information. Furthermore, when the rates of synonymous substitutions were included in the analysis of the E. coli data, only the variable expression levels remained statistically significant. The rate of nonsynonymous substitution was shown to correlate with expression levels independently of the rate of synonymous substitution. These results suggest an important direct influence of expression levels, or at least codon usage bias for translation optimization, on the rates of nonsynonymous substitutions in bacteria. They also indicate that when a control for this variable is included, essentiality plays no significant role in the rate of protein evolution in bacteria, as is the case in eukaryotes.     

A prevalent POLG CAG microsatellite length allele in humans and African great apes

The human nuclear gene for the catalytic subunit of mitochondrial DNA polymerase (POLG) contains within its coding region a CAG microsatellite encoding a polyglutamine repeat. Previous studies demonstrated an association between length variation at this repeat and male infertility, suggesting a mechanism whereby the prevalent (CAG)₁₀ allele, which occurs at a frequency of >80% in different populations, could be maintained by selection. Sequence analysis of the POLG CAG microsatellite region of more than 1000 human chromosomes reveals that virtually all allelic variation at the locus is accounted for by length variation of the CAG repeat. Analysis of POLG from African great apes shows that a prevalent length allele is present in each species, although its exact length is species-specific. In common chimpanzee (Pan troglodytes) a number of different sequence variants contribute to the prevalent length allele, strongly supporting the idea that the length of the POLG microsatellite region, rather than its exact nucleotide or amino acid sequence, is what is maintained. Analysis of POLG in other primates indicates that the repeat has expanded from a shorter, glutamine-rich sequence, present in the common ancestor of Old and New World monkeys.     

From NMR chemical shifts to amino acid types: Investigation of the predictive power carried by nuclei

An approach to automatic prediction of the amino acid type from NMR chemical shift values of its nuclei is presented here, in the frame of a model to calculate the probability of an amino acid type given the set of chemical shifts. The method relies on systematic use of all chemical shift values contained in the BioMagResBank (BMRB). Two programs were designed, one (BMRB stats) for extracting statistical chemical shift parameters from the BMRB and another one (RESCUE2) for computing the probabilities of each amino acid type, given a set of chemical shifts. The Bayesian prediction scheme presented here is compared to other methods already proposed: PROTYP RESCUE and PLATON and is found to be more sensitive and more specific. Using this scheme, we tested various sets of nuclei. The two nuclei carrying the most information are C(beta) and H(beta), in agreement with observations made in Grzesiek and Bax, 1993. Based on four nuclei: H(beta), C(beta), C(alpha) and C', it is possible to increase correct predictions to a rate of more than 75%. Taking into account the correlations between the nuclei chemical shifts has only a slight impact on the percentage of correct predictions: indeed, the largest correlation coefficients display similar features on all amino acids.     

DNA modification by oxovanadium(IV) complexes of salen derivatives

Oxovanadium(IV) complexes of hydroxysalen derivatives have been prepared and tested as DNA reactive agents. The nuclease activity has been investigated under oxidative or reducing conditions, on the basis of the various oxidation states of vanadium: V^III, V^IV and V^V. In the absence of an activating agent, none of the compounds tested was able to induce cleavage of DNA, whereas in the presence of mercaptopropionic acid (MPA) or Oxone the four complexes induced DNA modifications. Under both conditions, the para-hydroxy complex was found to be the most active compound. Reaction of these salen complexes with DNA occurs essentially at guanine residues and is more efficient in the presence of Oxone than under reducing conditions. The extent of Oxone-mediated DNA oxidation by the four vanadyl complexes was clearly superior to VOSO₄ and was observed without piperidine treatment. EPR studies provided information on the reactive metal-oxo species involved under each conditions and a mechanism of reaction with DNA is discussed.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0529-0Abbreviations BPE buffer bis-phosphate EDTA buffer - DMPO 5,5-dimethylpyrroline N-oxide - DMS dimethyl sulfate - HFS hyperfine structure - Lin linear - MPA 3-mercaptopropionic acid - Nck nicked - salen (salicylidene)ethylenediamine - Sc supercoiled - TBE buffer tris-borate EDTA buffer - Tris tris(hydroxymethyl)aminomethane     

HPLC-PAD-APCI/MS assay of phenylpropanoids in cereals

A new, rapid HPLC-PAD-APCI/MS assay has been developed in order to measure accurately the amount of p-coumaric, E- and Z-ferulic acid and the dehydrodimers of ferulic acid in cereal grain. In the positive ionisation mode, MS patterns gave additional information for the identification of the dimers. The time required and the quantities of solvents employed in the developed analytical method are much lower than those involved in previously available assays of these compounds, thus making the method suitable for the screening of cereal genotypes. Application of the method to accessions of maize, wheat and sorghum showed that E-ferulic was the most abundant phenylpropanoid, whilst the major dimer was 8-O-4' dehydrodimer of ferulic acid followed by the 5-5' and then the 8-5' forms. Maize grains, especially of the Mexican landraces, contained the highest levels of these dimers.     

Endocytosis by random initiation and stabilization of clathrin-coated pits

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have detected the formation of coats by monitoring incorporation of fluorescently tagged clathrin or its adaptor AP-2; we have also followed clathrin-mediated uptake of transferrin and of single LDL or reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no strongly preferred nucleation sites. A proportion of the nucleation events are weak and short lived. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit. Our data lead to a model in which coated pits initiate randomly but collapse unless stabilized, perhaps by cargo capture.     

AliasServer: a web server to handle multiple aliases used to refer to proteins

SUMMARY: AliasServer provides services that facilitate the assembly of data or datasets that make use of different identifiers for refering to the same protein. This resource relies on a database which contains, for a given organism, a non-redundant list of protein sequences associated with a set of aliases. AVAILABILITY: AliasServer is available as an interactive Web server at http://cbi.labri.fr/outils/alias/ and as a web service using a SOAP interface. The complete tool, including sources and data, is available for local installations upon request. SUPPLEMENTARY INFORMATION: Technical documentation is available at http://cbi.labri.fr/outils/alias/asdoc.pdf