Directed search for host surface proteins as antiviral targets against SARS-CoV-2

In late 2019 a novel coronavirus emerged in the human population in Hubei province, China from an unknown animal source. In four months, it spread to 190 countries and as of March 24th it infected more than 300'000 people worldwide, 15'000 of which with fatal outcome. Governments all over the world struggle to contain the pandemic, with never before experienced population-wide lockdown measures. Socio-economic consequences of this lockdown are not foreseeable yet. The virus, named SARS-CoV-2, is closely related to the 2002 SARS-CoV-1 and causes mild to severe respiratory symptoms and in severe cases viral pneumonia and death. Especially elderly patients are at high risk of severe disease progression with an elevated chance of mortality (15%) in consequence of coronavirus disease 19 (COVID19). Currently no vaccine is available and repurposing of antiviral drugs is still in an early stage. Thus, therapy is currently largely limited to support measures like oxygen and ventilation, if required. To address the Swiss National Fund Special Call: Coronavirus, we here propose a project, aiming to identify novel antiviral targets on the host cell surface. As all viruses, SARS-CoV-2 depends on host proteins for its replication. This affects every step of the viral replication cycle, including viral entry. As a bona fide receptor for entry ACE2 was identified, but cofactors of this receptor are currently not known. We propose here a strategy, which was already successfully tested to identify influenza A virus hemagglutinin host interaction partners on the surface of human lung epithelial cells, which were then targeted by chemical compounds to successfully reduce viral replication. Our strategy is based on soluble viral glycoproteins (S protein for SARS-CoV-2) fused to HRP. This fusion protein is stabilized by a trimerization domain to achieve the natural trimeric confirmation of the S-protein ectodomain. We will use insect cell produced SARS-CoV-2 S-protein-HRP as molecular bait, which, when attached to the host cell surface, mark host proteins in close vicinity by biotin ligation (proximity ligation assay). Biotinylated host surface proteins will be identified by mass spectrometry after streptavidin pulldown. Their role in early viral replication events will be confirmed functionally by genetic (siRNA or CRISPR/Cas9) or chemical targeting followed by infection with VSV-?G, pseudo-typed with SARS-CoV-2 S-protein. Our strategy is a directed approach to broaden the spectrum of antiviral targets, urgently needed in the development of novel antivirals to mitigate the current SARS-CoV-2 pandemic.