Journal: Nature Communications

Article Title: Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

doi: 10.1038/s41467-023-38717-w

Figure Lengend Snippet: Summary statistics for novel coronavirus genomes assembled in this study

Article Snippet: It consists of a gene synthesised by Genscript of CHO codon-optimised sequence of RhGB07, residues 1–1191, preceded by a u-phosphatase signal peptide , residues 969 and 970 mutated to proline (2 P) to stabilise the prefusion state of the spike trimer, a putative basic site that may be the site of proteolysis (RAKQ, residues 669–672, was mutated to GASQ), a C-terminal T4 foldon fusion domain to stabilise the trimer complex, followed by C-terminal 8x His and 2x Strep tags for affinity purification.

Techniques:

Journal: Nature Communications

Article Title: Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

doi: 10.1038/s41467-023-38717-w

Figure Lengend Snippet: a Entry of different spike pseudoviruses expressing viral glycoproteins into HEK293T cells transfected with ( a ) human receptors known to allow entry of human coronaviruses or ( e ) ACE2 homologues from different species. a , e The raw entry values for each pseudoviruses were normalised by their entry into cells transfected with a vector containing no receptor sequence (i.e., “empty”). The raw entry values of representative repeats ( n = 3 independent experiments) are also shown for direct comparisons of absolute entry. Representative biolayer interferometry binding curves showing the association and dissociation of SARS-CoV-2 and RhGB07 spike proteins with ( b ) hACE2 ( n = 2 and 3 independent experiments, at three and seven protein concentrations, respectively), or ( f ) with R. ferrumequinum or M. lucifugus ACE2 ( n = 1 independent experiment, at seven protein concentrations). c Entry of pseudoviruses into different “normal” human-cell lines that stably express lower or physiological levels of hACE2. All entry measurements are normalised to those for the “bald” pseudovirus not expressing any spike protein. d Entry of pseudoviruses into Huh7.5 cells transduced with a human TMPRSS2 vector, normalised to “bald”. Data from panels ( a , c – e ) are compiled from n = 3–8 independent experiments and plotted as mean + s.d. Statistical significance was determined by ( a , e ) two-way ANOVA or c , d one-way ANOVA on log-transformed data (after determining log normality by the Shapiro–Wilk test and QQ plot) with multiple comparisons against “empty” vector or “bald” pseudovirus, respectively. *0.05 ≥ P > 0.01; **0.01 ≥ P > 0.001; ***0.001 ≥ P > 0.0001; **** P ≤ 0.0001. Exact P values annotated for each graph are as follows (from left to right), ( a ) <0.0001, <0.0001, <0.0001, <0.0001; ( c , Calu-3) < 0.0001, <0.0001, 0.001, <0.0001, <0.0001; ( c , Caco-2) < 0.0001, <0.0001, 0.022, <0.0001, <0.0001; ( c , HEK293T-ACE2) < 0.0001, <0.0001, <0.0001; d <0.0001, <0.0001, <0.0001. ( e , SARS-CoV-2) < 0.0001, 0.0003, 0.12, <0.0001, <0.0001 ( e , BANAL-20-52) < 0.0001, <0.0001, <0.0001, <0.0001, <0.0001; ( e , RatG13) < 0.0001, <0.0001, <0.0001, <0.0001 ( e , RhGB07) < 0.0001, <0.0001; ( e , RfGB02) < 0.0001.

Article Snippet: It consists of a gene synthesised by Genscript of CHO codon-optimised sequence of RhGB07, residues 1–1191, preceded by a u-phosphatase signal peptide , residues 969 and 970 mutated to proline (2 P) to stabilise the prefusion state of the spike trimer, a putative basic site that may be the site of proteolysis (RAKQ, residues 669–672, was mutated to GASQ), a C-terminal T4 foldon fusion domain to stabilise the trimer complex, followed by C-terminal 8x His and 2x Strep tags for affinity purification.

Techniques: Expressing, Transfection, Plasmid Preparation, Sequencing, Binding Assay, Stable Transfection, Transduction, Transformation Assay

Journal: Nature Communications

Article Title: Genomic screening of 16 UK native bat species through conservationist networks uncovers coronaviruses with zoonotic potential

doi: 10.1038/s41467-023-38717-w

Figure Lengend Snippet: a The solved RBD structures of SARS-CoV-2, RaTG13, BANAL-236 (close relative of BANAL-20-52 ) and the AlphaFold2-predicted structure of RhGB07 were superposed. b The 3D surfaces of the RBD-hACE2 binding interface for SARS-CoV-2 and RhGB07. Alignment of sarbecovirus spike proteins showing the conservation of key contact residues involved interactions between ( c ) SARS-CoV spike and ( d ) SARS-CoV-2 spike with hACE2. The sequences shown in the alignments are from Asian, European and African sarbecoviruses that have been shown to bind hACE2 – . These sequences were ordered based on their genetic relatedness, as inferred from a consensus maximum-likelihood phylogenetic tree reconstructed from their whole genomes (bottom left).

Article Snippet: It consists of a gene synthesised by Genscript of CHO codon-optimised sequence of RhGB07, residues 1–1191, preceded by a u-phosphatase signal peptide , residues 969 and 970 mutated to proline (2 P) to stabilise the prefusion state of the spike trimer, a putative basic site that may be the site of proteolysis (RAKQ, residues 669–672, was mutated to GASQ), a C-terminal T4 foldon fusion domain to stabilise the trimer complex, followed by C-terminal 8x His and 2x Strep tags for affinity purification.

Techniques: Binding Assay