SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha

Time-resolved fluorescence of 2-aminopurine in DNA duplexes in the presence of the EcoP15I Type III restriction-modification enzyme

AbstractEcoP15I is a Type III DNA restriction and modification enzyme of Escherichia coli. We show that it contains two modification (Mod) subunits for sequence-specific methylation of DNA and one copy of a restriction endonuclease (Res) subunit for cleavage of DNA containing unmethylated target sequences. Previously the Mod2 dimer in the presence of cofactors was shown to use nucleotide flipping to gain access to the adenine base targeted for methylation (Reddy and Rao, J. Mol. Biol. 298 (2000) 597–610.). Surprisingly the Mod2 enzyme also appeared to flip a second adenine in the target sequence, one which was not subject to methylation. We show using fluorescence lifetime measurements of the adenine analogue, 2-aminopurine, that only the methylatable adenine undergoes flipping by the complete Res1Mod2 enzyme and that this occurs even in the absence of cofactors. We suggest that this is due to activation of the Mod2 core by the Res subunit

Preferential bonding in low alkali borosilicate glasses

International audienceIn an earlier review we discussed the connectivity in borosilicate glasses and compared our experimental findings by NMR, infrared and Raman spectroscopies with older structural models. We could show, contrary to the often cited reedmergnerite type model, that a significant preference exists in low alkali borosilicate glasses for trigonal rather than tetrahedral borate groups to link to silicate entities. Another often cited misconception is the application of the Loewenstein rule to borate tetrahedra. While linking of two "[AlØ4]– tetrahedra is disadvantageous compared to higher coordinated aluminate polyhedra, the borate tetrahedral units represents already the alternate higher coordination state and accordingly, many examples of linked [BØ4]– tetrahedra are known to exist in glasses as well as in crystalline compounds of boron at normal pressure conditions. We now present more NMR data on three different low alkali borosilicate glasses with Na2O:B2O3=0·2 to 0·35 and decreasing SiO2 fractions (74 to 43 mol% SiO2), for which we compare variations in the near and intermediate range structure of quenched and slowly annealed samples. None of the studied glasses showed a significantly higher fraction of trigonal BØ3 groups in the quenched than in the annealed samples, even though borate in the three-coordinated state is the preferred metaborate unit in the melt. However, for the two silicate rich glasses (including NBS 2), we observe at low temperatures a deviation of the viscosity–temperature plot from the ideal VFT-fit, that is from Tg at circa 440 to the expected 600°C. For samples prepared at any annealing temperature below 600°C, structural variations with thermal history are apparent, and are also reflected in many glass properties including density, fracture probability, or refractive index. Even though the glass NBS 2 shows no visible phase separation, DSC measurements indicate the presence of two different Tg values corresponding to the borate and the silica rich subnetworks. The values of the two Tg events shift with different cooling rates: for fast quenched glasses the two Tg values are closer together than for slowly annealed glasses. The study of these low alkali borosilicate glasses is ongoing, as we understand better how structural variations with changing thermal history impact the glasses' properties. Analogously, we can apply the same techniques to follow structural variations under external forces, such as irradiation, laser modification, pressure and mechanical impact