Screening and testing for a suitable untransfected cell line for SARS-CoV-2 studies

Abstract

<jats:title>Abstract</jats:title><jats:p>At present, the novel pandemic coronavirus SARS-CoV-2 is a major global threat to human health and hence demands united research activities at different levels. Finding appropriate cell systems for drug screening and testing molecular interactions of the virus with the host cell is mandatory for drug development and understanding the mechanisms of viral entry and replication. For this, we selected human cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) based on RNA-seq data determined transcript levels of ACE2 and TMPRSS2, two membrane proteins that have been identified to aid SARS-CoV-2 entry into the host cell. mRNA and protein expression of these host factors were verified via RQ-PCR and western blot. We then tested permissiveness of these cell lines towards SARS-CoV-2 infection, cytopathic effect, and viral replication finding limited correlation between receptor expression and infectability. One of the candidate cancer cell lines, the human colon cancer cell line CL-14, tested positive for SARS-CoV-2 infection. Our data argue that SARS-CoV-2 in vitro infection models need careful selection and validation since ACE2/TMPRSS2 receptor expression on its own does not guarantee permissiveness to the virus.</jats:p><jats:sec><jats:title>Author summary</jats:title><jats:p>In the midst of the pandemic outbreak of corona-virus SARS-CoV-2 therapeutics for disease treatment are still to be tested and the virus-host-interactions are to be elucidated. Drug testing and viral studies are commonly conducted with genetically manipulated cells. In order to find a cell model system without genetic modification we screened human cell lines for two proteins known to facilitate entry of SARS-CoV-2. We confirmed and quantified permissiveness of current cell line infection models, but dismissed a number of receptor-positive cell lines that did not support viral replication. Importantly, ACE2/TMPRSS2 co-expression seems to be necessary for viral entry but is not sufficient to predict permissiveness of various cancer cell lines. Moreover, the expression of specific splice variants and the absence of missense mutations of the host factors might hint on successful infection and virus replication of the cell lines.</jats:p></jats:sec>

SARS-CoV-2 hijacks macropinocytosis to facilitate its entry and promote viral spike–mediated cell-to-cell fusion

“…Since TMPRSS2 can mediate the direct fusion of viral and plasma membranes in several cell lines [7,10], we next tested whether the above tested pathways were involved in SARS-CoV-2 replication in TMPRSS2-expressing cells. Caco-2 cells have been demonstrated to express high levels of endogenous TMPRSS2 and have been widely used as a SARS-CoV-2 infection model (25,26). We found that all these inhibitors could inhibit SARS-CoV-2 replication in Caco-2 cells (Fig.…”