# The Introduction of a HuR-Binding Site in the 3′ UTR and the CD47 Cytoplasmic Tail Enhances SARS-CoV-2 S-Protein Expression in Cells

## TL;DR

This study shows that adding a HuR-binding site and a CD47 cytoplasmic tail to the SARS-CoV-2 S-protein increases its expression and surface presentation in cells.

## Contribution

The novel use of CD47 elements to enhance SARS-CoV-2 S-protein expression and surface localization is introduced.

## Key findings

- Deleting the 19 C-terminal amino acids of the S-protein increased its cell surface presentation.
- Adding a HuR-binding site and CD47 cytoplasmic tail significantly enhanced total S-protein expression.
- Higher surface expression with CD47 elements correlated with increased total protein levels.

## Abstract

In this study, we constructed plasmids to increase the overall expression level of the SARS-CoV-2 S-protein and its presentation on the cell surface. To this end, we designed a series of plasmid constructs encoding the SARS-CoV-2 S-protein with modifications to its cytoplasmic domain and containing various 5′ and 3′ untranslated regions. Our results confirmed the critical role of the S-protein cytoplasmic domain in limiting its localization to the cell surface. We confirmed that deletion of the 19 C-terminal amino acids, which contain an endoplasmic reticulum retrieval signal, significantly increased S-protein presentation on the cell surface. Furthermore, introducing the HuR-binding site from the CD47 3′ untranslated region and replacing the 19 C-terminal amino acids of the S-protein with the CD47 cytoplasmic tail significantly enhanced total S-protein expression compared to the wild-type S-protein and constructs with the 19-amino-acid deletion. Unfortunately, for the plasmid constructs bearing CD47 elements, their higher surface expression compared to the wild-type S-protein correlated with a high total protein expression level.