Structure, Function, and Anti-Viral Inhibition of the SARS-CoV-2 Envelope Protein Ion Channel
Kiefer Ramberg,a,b Nikitas Ostrovitsa,c Samir Olatunji,a Lysbeth Antonides,b Quenton Hurst,d Darya Marchany Rivera,e Honor Glenn,e Liqiang Zhang,e Samantha Pitt,d Eoin Scanlan,b Ulrich Zachariae,b Brenda G. Hoguee and Martin Caffreya
aSchool of Biochemistry & Immunology, Trinity College Dublin, Ireland.
bSchool of Life Sciences, University of Dundee, UK.
cSchool of Chemistry, Trinity College Dublin, Ireland.
dSchool of Medicine, University of St Andrews, UK.
eSchool of Life Sciences, Arizona State University, USA.
While the SARS-CoV-2 vaccination effort is successfully preventing severe cases of COVID-19 and death in developed countries, antiviral inhibitors are still in demand. Most efforts have focused on targeting the viral spike protein to prevent viral adhesion to cells. Another approach is to target SARS-CoV-2 structural proteins involved in viral assembly and release. The SARS-CoV-2 envelope (E) protein is a small (75 residue) integral membrane protein which functions as a pentameric viroporin and is implicated in viral budding, the release of progeny viruses, and activation of host inflammasomes. It is now widely agreed that this inflammasome activation upon infection by the SARS-CoV-2 pathogen is responsible for the hyperinflammatory response (cytokine storm) that accompanies onset of severe disease. Inhibition of E-protein ion channel has been shown to impede viral assembly and release of viral particles. To date, structural studies of E have been limited to NMR investigations, with significant disparity between different structural models. High resolution crystal structures would address these disparities and allow for evaluation of drug binding sites on E and identification of potential antiviral inhibitors. To this end, we are pursuing E crystallization in the lipidic cubic phase, a membrane mimetic widely implemented for the crystallization of important drug targets such as GPCRs. Crystallization trials with two E protein constructs are underway: the full-length protein produced recombinantly (EFL, 1-75) and the transmembrane domain produced via solid phase peptide synthesis (ETM, 8-38). Concurrently, we are pursuing functional characterization of E via single molecule planar lipid bilayer electrophysiology and a liposome dye release assay. Furthermore, we are studying the lipid interactions of the E protein as well as persistent pore formation and conductivity through molecular dynamic simulations.

**Structure, Function, and Anti-Viral Inhibition of the SARS-CoV-2 Envelope Protein Ion Channel**
Kiefer Ramberg,^a,b^ Nikitas Ostrovitsa,^c^ Samir Olatunji,^a^ Lysbeth Antonides,^b^ Quenton Hurst,^d^ Darya Marchany Rivera,^e^ Honor Glenn,e Liqiang Zhang,^e^ Samantha Pitt,^d^ Eoin Scanlan,^b^ Ulrich Zachariae,^b^ Brenda G. Hogue^e^ and Martin Caffrey^a^
^a^School of Biochemistry & Immunology, Trinity College Dublin, Ireland.
^b^School of Life Sciences, University of Dundee, UK.
^c^School of Chemistry, Trinity College Dublin, Ireland.
^d^School of Medicine, University of St Andrews, UK.
^e^School of Life Sciences, Arizona State University, USA.
While the SARS-CoV-2 vaccination effort is successfully preventing severe cases of COVID-19 and death in developed countries, antiviral inhibitors are still in demand. Most efforts have focused on targeting the viral spike protein to prevent viral adhesion to cells. Another approach is to target SARS-CoV-2 structural proteins involved in viral assembly and release. The SARS-CoV-2 envelope (E) protein is a small (75 residue) integral membrane protein which functions as a pentameric viroporin and is implicated in viral budding, the release of progeny viruses, and activation of host inflammasomes. It is now widely agreed that this inflammasome activation upon infection by the SARS-CoV-2 pathogen is responsible for the hyperinflammatory response (cytokine storm) that accompanies onset of severe disease. Inhibition of E-protein ion channel has been shown to impede viral assembly and release of viral particles. To date, structural studies of E have been limited to NMR investigations, with significant disparity between different structural models. High resolution crystal structures would address these disparities and allow for evaluation of drug binding sites on E and identification of potential antiviral inhibitors. To this end, we are pursuing E crystallization in the lipidic cubic phase, a membrane mimetic widely implemented for the crystallization of important drug targets such as GPCRs. Crystallization trials with two E protein constructs are underway: the full-length protein produced recombinantly (EFL, 1-75) and the transmembrane domain produced via solid phase peptide synthesis (ETM, 8-38). Concurrently, we are pursuing functional characterization of E via single molecule planar lipid bilayer electrophysiology and a liposome dye release assay. Furthermore, we are studying the lipid interactions of the E protein as well as persistent pore formation and conductivity through molecular dynamic simulations.

Structure, Function, and Anti-Viral Inhibition of the SARS-CoV-2 Envelope Protein Ion Channel

Exploring the intricacies of host-pathogen communication is vital in order to understand why
some microbes persist within a host, while others are effectively cleared. As such,
host-pathogen communication is an urgent area of research underpinning human health
and disease, especially given the emerging concerns of antibiotic resistance.
Understanding host-pathogen pathways requires intricate knowledge in the fields of both
microbiology and immunology.

Living in a world of specialists, where scientists sometimes invest their whole career
studying one organism, a specific pathway or a protein, multidisciplinary discussions of
host-pathogen interactions are highly needed. This international conference will provide
such a forum for scientific exchange at the highest level, between scientists from different
research areas related to host-pathogen interactions, with the aim to trigger fruitful
collaborations addressing the urgent questions in the field.

This meeting has an additional special focus of bringing researchers from Ireland, Denmark
and Israel together to learn about cutting edge biology and form new collaborations.
Thus, the conference will be a showcase for the exciting and original research emanating
from these three small but innovative countries.