The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.

Significance: The COVID-19 pandemic caused by SARS-CoV-2 infection has led to more than 840,000 deaths worldwide as of August 31, 2020. Unfortunately, no licensed vaccine or specific treatment is available right now. Herein, we report a decoy nanoparticle against COVID-19. The decoy nanoparticles were constructed by fusing cell membrane nanovesicles derived from genetically engineered cells, which stably express SARS-CoV-2 receptor ACE2, and human monocytes, which display abundant cytokine receptors. By competing with host cells, these nanodecoys efficiently adsorb viruses and inflammatory cytokines such as IL-6 and GM-CSF. These two functionalities allow effective intervention of viral infection and its associated immune disorder, presenting a promising therapeutic strategy for COVID-19 and other potential epidemics.