# C-terminus-outward orientation of SARS-CoV-2 envelope proteins on viral capsid enables a novel virus–cell interaction pathway

## TL;DR

This study shows that the C-terminus of the SARS-CoV-2 envelope protein is on the outside of the virus and can interact with host cells, suggesting a new pathway for viral infection.

## Contribution

The study reveals the C-terminus-outward orientation of SARS-CoV-2 envelope proteins and their role in host cell interaction.

## Key findings

- The C-terminal half of the E-pr is exposed on the surface of the SARS-CoV-2 virus.
- EC38 peptides modulate gene expression in human vascular endothelial cells, affecting hemostasis and blood pressure regulation.
- EC38 interacts with proteins involved in cell adhesion, cytoskeleton organization, and immune signaling.

## Abstract

Since the onset of COVID-19, emphasis has been largely placed on spike proteins, while other molecules of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), such as the 78-aa-long envelope protein (E-pr), have been largely overlooked. This study was conducted to identify the orientation of E-pr in the envelope of the living SARS-CoV-2 virus and to confirm whether the viroid E-pr plays a role in the attachment and stimulation of the host cells. Using a customized antisera against the C-terminal half of E-pr (EC38, 38 aa), we demonstrated that the EC38 segment was located outside of the intact virus. Synthetic peptides with the sequence of the EC38 segment were bound to and modulated gene expression in cultured human vascular endothelial cells (HUVECs), and altered genes were enriched for the ontology terms “hemostasis” and “regulation of systemic arterial blood pressure by hormone.” EC38-mediated pull-down of HUVEC membrane proteins plus mass spectrometry revealed that the EC38-interacting proteins were enriched for “actin cytoskeleton organization,” “cell–cell interaction,” “cell–cell adhesion,” “hemostasis,” “viral infection pathways,” “cytokine signaling in immune system,” etc., among which CKAP4, vimentin, and ATP5B were further identified to bind EC38 peptides. Molecular docking supported the potential binding of the E-pr with the ATP synthase. In summary, by displaying a C-terminal-outward orientation on the surface of the SARS-CoV-2 virus, E-pr was able to adhere to and stimulate host cells. Future studies should address whether the E-pr pathway could be utilized as a target for managing SARS-CoV-2 infection in humans.