SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis

Armin Bayati,Rahul Kumar,Vincent Francis,Peter S Mcpherson

doi:10.1016/j.jbc.2021.100306

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity.

Highlights

Since the beginning of the 21st century, 3 coronaviruses have crossed the species barrier to cause deadly types of pneumonia in humans: Middle-East respiratory syndrome coronavirus (MERS-CoV) [1], severe acute respiratory syndrome coronavirus (SARS-CoV) [2, 3], and SARS-CoV-2 [4, 5], the causative agent of COVID-19
SARS-CoV-2 spike glycoprotein is rapidly endocytosed in an angiotensin-converting enzyme 2 (ACE2)-dependent manner
The spike glycoprotein is critical for binding ACE2 and allowing for SARS-CoV-2 infectivity [6, 8,9,10,11,12, 16, 17]

Summary

Introduction

Since the beginning of the 21st century, 3 coronaviruses have crossed the species barrier to cause deadly types of pneumonia in humans: Middle-East respiratory syndrome coronavirus (MERS-CoV) [1], severe acute respiratory syndrome coronavirus (SARS-CoV) [2, 3], and SARS-CoV-2 [4, 5], the causative agent of COVID-19. Endocytosis assay using purified spike protein Cells were incubated at 37 C with serum-free media (DMEM) for 3 h to enhance ACE2 receptor expression. HEK-293T cells or HEK-293T cells stably expressing ACE2 [30] were incubated for 30 min on ice with purified spike protein containing a His6 tag. After a PBS wash, spike protein binds to the plasma membrane of cells expressing ACE2 but not to the control HEK-293T cells (Fig. 1, A and B).

Results

Conclusion