Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration.

ABSTRACT

The Spike protein of SARS-CoV-2, its receptor-binding domain (RBD), and its primary receptor ACE2 are extensively glycosylated. The impact of this post-translational modification on viral entry is yet unestablished. We expressed different glycoforms of the Spike-protein and ACE2 in CRISPR-Cas9 glycoengineered cells, and developed corresponding SARS-CoV-2 pseudovirus. We observed that N- and O-glycans had only minor contribution to Spike-ACE2 binding. However, these carbohydrates played a major role in regulating viral entry. Blocking N-glycan biosynthesis at the oligomannose stage using both genetic approaches and the small molecule kifunensine dramatically reduced viral entry into ACE2 expressing HEK293T cells. Blocking O-glycan elaboration also partially blocked viral entry. Mechanistic studies suggest multiple roles for glycans during viral entry. Among them, inhibition of N-glycan biosynthesis enhanced Spike-protein proteolysis. This could reduce RBD presentation on virus, lowering binding to host ACE2 and decreasing viral entry. Overall, chemical inhibitors of glycosylation may be evaluated for COVID-19.

COVID-19 is an infectious disease caused by the virus SARS-CoV-2. To access the internal machinery necessary for its replication, the virus needs to latch onto and then enter host cells. Such processes rely on specific 'glycoproteins' that carry complex sugar molecules (or glycans), and can be found at the surface of both viruses and host cells. In particular, the viral 'Spike' glycoprotein can attach to human proteins called ACE2, which coat the cells that line the inside of the lungs, heart, kidney and brain. Yet the roles played by glycans in these processes remains unclear. To investigate the role of Spike and ACE-2 glycans, Yang et al. designed a form of SARS-CoV-2 that could be handled safely in the laboratory. How these viruses infect human kidney cells that carry ACE2 was then examined, upon modifying the structures of the sugars on the viral Spike protein as well as the host ACE2 receptor. In particular, the sugar structures displayed by the virus were modified either genetically or chemically, using a small molecule that disrupts the formation of the glycans. Similar methods were also applied to modify the glycans of ACE2. Together, these experiments showed that the sugars present on the Spike protein play a minor role in helping the virus stick to human cells.However, they were critical for the virus to fuse and enter the host cells. These findings highlight the important role of Spike protein sugars in SARS-CoV-2 infection, potentially offering new paths to treat COVID-19 and other coronavirus-related illnesses. In particular, molecules designed to interfere with Spike-proteins and the viral entrance into cells could be less specific to SARS-CoV-2 compared to vaccines, allowing treatments to be efficient even if the virus changes.