Choosing a cellular model to study SARS-CoV-2

Abstract

As new pathogens emerge, new challenges must be faced. This is no different in infectious disease research, where identifying the best tools available in laboratories to conduct an investigation can, at least initially, be particularly complicated. However, in the context of an emerging virus, such as SARS-CoV-2, which was recently detected in China and has become a global threat to healthcare systems, developing models of infection and pathogenesis is urgently required. Cell-based approaches are crucial to understanding coronavirus infection biology, growth kinetics, and tropism. Usually, laboratory cell lines are the first line in experimental models to study viral pathogenicity and perform assays aimed at screening antiviral compounds which are efficient at blocking the replication of emerging viruses, saving time and resources, reducing the use of experimental animals. However, determining the ideal cell type can be challenging, especially when several researchers have to adapt their studies to specific requirements. This review strives to guide scientists who are venturing into studying SARS-CoV-2 and help them choose the right cellular models. It revisits basic concepts of virology and presents the currently available in vitro models, their advantages and disadvantages, and the known consequences of each choice.

Keywords: COVID-19; SARS-CoV-2; cell lines; cell model; in vitro approaches; organoids; susceptible cells; viral culture.

Figure 1

SARS-Cov-2 Replication Cycle and antiviral restriction factors in each step of viral replication. (1) Adhesion: SARS-CoV-2 Spike (S) protein binds to a cellular receptor, which is mostly Angiotensin-Converting Enzyme 2 (ACE2), although alternative receptors are described (Ex: ASGR1, KREMEN1). (2) Entry: When there is expression of transmembrane protease serine 2 (TMPRSS2), this protease cleaves the viral Spike protein mediating entry by fusion of the viral membrane to the host cell membrane. In parallel, in the absence of expression of this protease, entry occurs by (2’) endocytosis mediated by the receptor, triggering the formation of endo-lysosomes in which Cathepsin L (CTSL) will be responsible for the cleavage of the Spike protein. A new conformational arrangement is induced by this cleavage, triggering (3) the viral genome (+ssRNA) release (via uncoating) into the cell cytoplasm. After the viral RNA is delivered into the host cell, the (4) translation of the viral replication machinery begins: the coronavirus genomic RNA encodes nonstructural proteins (NSPs) that have a critical role in (5) Viral Genome replication: process in which the virus induces the synthesis its RNA, mediated by NSPs. (6) Translation of Viral Structural Protein: the structural proteins S, Envelope (E), and Membrane (M) are translated by ribosomes that are bound to the endoplasmic reticulum (ER). The nucleocapsid proteins (N) remain in the cytoplasm and are assembled from genomic RNA. They fuse with the precursor virion, which is then transported from the ER through the Golgi Apparatus. The Spike cleavage at the S1/S2 furin site probably takes place when virions are released through the Golgi apparatus, responsible for the (7) Viral release: transporting virions to the cell surface via small vesicles, finally released by (8) Exocytosis: the viral progeny is released by exocytosis to the extracellular medium, ready to find and infect new cells. Many of these steps are antagonized by intact cell defense mechanisms, known as restriction factors, which stop viral replication in response to infection.

Figure 2

Comparison of SARS-CoV-2 permissive cell lines. Four human cell lines: Calu-3 (Pulmonary), Caco-2 (Intestinal), Huh-7 (Hepatic) and HEK 293T (Renal), are compared with each other and with the Vero E6 cell, a cell derived from the African green monkey kidney, and widely used in the isolation and production of SARS-CoV-2. Cells are compared for the expression of three important entry factors used by SARS-CoV-2: angiotensin-converting enzyme 2 receptor (ACE2), transmembrane protease serine 2 (TMPRSS2) and the lysosomal protease cathepsin L (CTSL), based on data from Murgolo et al., 2021; Saccon et al., 2021 and Shuai et al., 2020. The expression of these factors dictates the entry pathway used by SARS-CoV-2. The release of SARS-CoV-2 viral particles is also compared. The asterisks draw attention to likely consequences of producing SARS-CoV-2 in these cells.

Figure 3

Cell lines transfected to express or overexpress ACE2 and TMPRSS2 show increased isolation of, susceptibility to, and permissibility for SARS-CoV-2. A549 (Pulmonary) cell line has poor expression of ACE2 and no expression of TMPRSS2, being considered a non-permissive cell to SARS-CoV-2. When ACE2 or both components are expressed after transfection of the cell, it becomes moderately permissive and susceptible to SARS-CoV-2. HeLa cells (Uterine cervix) are not susceptible to SARS-CoV-2 infection, as they do not express the viral receptors. Transfection of ACE2 and TMPRSS2 leads to susceptibility and moderate permissibility. In susceptible cells, such as Vero E6 monkey cells and HEK 293T and human renal cells, transfection with the receptor which they do not express endogenously leads to greater cell permeability. Vero E6/TMPRSS2+ cells have higher rates of SARS-CoV-2 isolation and potentially prevent attenuation of the virus. Data concerning the expression profile of each cell were recovered from Vectorbuilder; Murgolo et al., 2021; Saccon et al., 2021; Shuai et al., 2020 and Hoffmann et al., 2020. The asterisk (*) draws attention to the fact that no data was found about the impact of the expression of only TMPRSS2 in A549 cells, but the combination of TMPRSS2 expression in A549 cells already modified to overexpress ACE2 leads to increased infectivity.

Figure 4

Schematic representation of a decision flowchart to choose which cell line to employ in SARS-CoV-2 studies. ACE2, angiotensin-converting enzyme 2 receptor; CPE, cytopathic effect; TMPRSS2, transmembrane protease serine 2; IFN1, Interferon type I.