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. 2021 Jun 17;22(12):6490.
doi: 10.3390/ijms22126490.

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

Olga A Postnikova  1 , Sheetal Uppal  1 , Weiliang Huang  2 , Maureen A Kane  2 , Rafael Villasmil  3 , Igor B Rogozin  4 , Eugenia Poliakov  1 , T Michael Redmond  1
Affiliations

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

Olga A Postnikova et al. Int J Mol Sci. .

Abstract

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.

Keywords: SARS-CoV-2; codon usage; ribosome pausing site; ribosome stalling; spike protein.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
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
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
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
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
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
Figure 6
COS7 infection with LV-produced pseudovirions. Experiments were conducted in duplicate and repeated several times. Scale bar = 180 μm.
Figure 7
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 7
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
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
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
Figure 10
Quantification of GFP expression by cytometry in Vero E6 cells following lentiviral infection. Error bars indicate the standard deviation.
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