Journal: Journal of Virology

Article Title: The “LLQY” Motif on SARS-CoV-2 Spike Protein Affects S Incorporation into Virus Particles

doi: 10.1128/jvi.01897-21

Figure Lengend Snippet: Tyrosine residue 756 is critical for the cleavage of the prefusion S protein. (A) Schematic of the domain structure of SARS-CoV-2 spike proteins and amino acid sequence alignment of the S1/S2 (R685) and S2′ (R815) cleavage site and the region between them in S2 subunit with the corresponding fragments of SARS-CoV and MERS-CoV. (B to D) The hydrogen bonds formed by Y756 and the leucine at position 753 (L753), the phenylalanine at position 759 (F759), or the threonine residue at position 998 (T998) in the folded S trimer can be observed in the cryo-EM structure of the S ectodomain trimer (PDB code 6VSB ). (E) Western blot analysis of the effect of Y756, R685, or R815 mutation on the expression and processing of SARS-CoV-2 spike proteins using anti-RBD (S1) and Flag (C-terminal of S2). GAPDH was used as a loading control. (F) Western blot analysis of the effect of Y756 mutation to different amino acids on the synthesis and processing of S protein using anti-RBD and anti-Flag antibodies. GAPDH was used as a loading control. (G) A summary diagram of the regulation of S protein cleavage when Y756 is mutated to phenylalanine (F), tryptophan (W), histidine (H), cysteine (C), alanine (A), glycine (G), or glutamate (E). (H) Western blot analysis of the effect of L753, F759, or T998 mutation on the synthesis and processing of S protein using anti-RBD and anti-Flag antibodies. GAPDH was used as a loading control.

Article Snippet: Rabbit polyclonal antibodies against Flag ( DYKDDDDK ) tag (no. 20543-1-AP), ACE2 (no. 21115-1-AP), GAPDH (no. 10494-1-AP), and β-actin (no. 20536-1-AP), and CoraLite 488-conjugated ACE2 monoclonal antibody (no. CL488-66699) were purchased from Proteintech (China).

Techniques: Residue, Sequencing, Cryo-EM Sample Prep, Western Blot, Mutagenesis, Expressing, Control