The Functional Consequences of the Novel Ribosomal Pausing Site in SARS-CoV-2 Spike Glycoprotein RNA

Figures

• 

  Figure 1. Fragment of the multiple alignments of SARS-CoV-2 S protein RNAs: Sequences surrounding the CCTCGGCGGGCA insertion in the SARS-CoV-2 sequence (GenBank entry NC_045512.2, the SARS-CoV-2 reference sequence). MN996532.1 is the closest bat homolog RaTG13; MG772934.1, MG772933.1, and KT444582.1 are more distantly related bat homologs. Novel out-of-frame stop codons in the human SARS-CoV-2 are italicized.

• 

  Figure 2. Expression of original and codon-optimized S proteins in HEK293F cell lysates. The left panel was imaged at normal gain, while the right panel was imaged at higher gain to visualize original S protein expression (S original). Experiments were repeated several times.

• 

  Figure 3. Expression of original and codon-optimized S proteins in Expi293 cell lysates. The left panel was imaged at normal gain, while the right panel was imaged at higher gain to visualize original S protein expression (S original). Experiments were repeated several times.

• 

  Figure 4. Expression of original and codon-optimized S proteins in HEK293T cell lysates. The left panel was imaged at normal gain, while the right panel was imaged at higher gain to visualize original S protein expression (S original). Experiments were repeated several times.

• 

  Figure 5. Expression of S protein in pseudovirions expressing original and codon-optimized S proteins. Pseudovirions were produced in LV production cells, Expi293 cells, and adherent HEK293T cells. Upper panels were imaged at normal gain, while lower panels were imaged at higher gain to visualize original S protein expression (S original). Red asterisk indicates spike protein band.

• 

  Figure 6. COS7 infection with LV-produced pseudovirions. Experiments were conducted in duplicate and repeated several times. Scale bar = 180 μm.

• 

  Figure 7. Infection of ARPE-19 cells with Expi293 produced pseudovirions: (A) GFP fluorescence analysis. Experiments were conducted in duplicate and repeated several times; scale bar = 180 μm; (B) %GFP-positive cells measured by Cytation7. Error bars indicate the standard deviation.

• 

  Figure 8. Vero E6 infection with HEK293T-produced pseudovirions measured by GFP fluorescence. Experiments were conducted in duplicate and repeated several times. Scale bar = 180 μm.

• 

  Figure 9. Vero E6 infection with LV-, Expi293-, and HEK293T-produced pseudovirions measured by GFP fluorescence in infected cells. Experiments were conducted in duplicate and repeated several times. Scale bar = 180 μm.

• 

  Figure 10. Quantification of GFP expression by cytometry in Vero E6 cells following lentiviral infection. Error bars indicate the standard deviation.