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.

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.

With more than 13 million cases and 570,000 deaths, and with the resulting social upheaval, the COVID-19 pandemic presents one of the greatest challenges ever to the scientific community. It is thus vital to fully understand the biology of SARS-CoV-2, the causative agent of COVID-19. SARS-CoV-2 uses the spike glycoprotein to interact with the cell surface and to drive fusion of the viral membrane with cellular membranes, thus allowing transfer of viral RNA to the cytosol. Here we use purified spike glycoprotein protein and lentivirus pseudotyped with spike glycoprotein to determine that SARS-CoV-2 undergoes rapid endocytosis following binding to the plasma membrane. Using chemical inhibitors and loss of function approaches, we demonstrate that this cellular entry is through clathrin-mediated endocytosis. Thus, it appears that SARS-CoV-2 first engages the plasma membrane, then rapidly enters the lumen of the endosomal system, strongly suggesting that fusion of the viral membrane occurs with the lumenal membrane of endosomes. This discovery has important implications for the development of chemical probes to reduce or block infection.

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