A Newly Engineered A549 Cell Line Expressing ACE2 and TMPRSS2 Is Highly Permissive to SARS-CoV-2, Including the Delta and Omicron Variants

Abstract

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Keywords: A549; ACE2; EIDD-1931; SARS-CoV-2; TMPRSS2; delta and omicron variants; nirmatrelvir; remdesivir.

Figure 1

Establishing a highly permissive ACE2plus cell line for SARS-CoV-2 infection. (A) Experimental scheme to establish A549-ACE2-TMPRSS2 (43.20) and A549-ACE2-TMPRSS2 (ACE2plus) cells. (B) Immunofluorescence staining of the spike protein of SARS-CoV-2 at 48 h post-infection. Scale bar, 100 μm. (C) ACE2plus cells show increased infectivity. The percentage of infected cells was quantified using ImageXpress to determine the infectivity (spike + cells in total cell number). Data are expressed as mean ± SD. n = 6 biological replicates. ****, p < 0.0001.

Figure 2

The characterization of single-cell clones from the ACE2plus cell population. (A) The experimental scheme to establish the ACE2plusC3 line. The commercial A549-hACE2-TMPRSS2 cell line (IVG-AT) was tested using the same experimental conditions. (B) Virus-infected cells are visualized by immunofluorescence staining of the nucleocapsid protein of SARS-CoV-2 at 48 h post-infection. Scale bar, 200 μm. Syncytia are indicated by asterisks. (C) The percent infectivity is determined by the number of NP+ (nucleocapsid protein) cells out of the total cells. The data are expressed as mean ± SD. n = 6 biological replicates. ****, p < 0.0001.

Figure 3

The characterization of the ACE2plus and ACE2plusC3 cell lines. The mRNA expression levels of ACE2 and TMPRSS2 in the indicated cell lines were measured with RT-qPCR (A,D). IVG-A and IVG-A/T commercial cell lines were used as comparators. (B) The cell surface ACE2 expression level was measured with flow cytometry using live cells. (C) The cell-doubling time was determined using a cell growth curve and Celigo Image software. (E) The protein expression levels of ACE2 and TMPRSS2 in cells were examined by immunoblotting and immunofluorescence staining (F). Scale bar, 100 μm. (G) The virus infectivity remains stable between early and late passages (p9, p12, p17, p20, and p23). ACE2plusC3 cells were infected with WT SARS-CoV-2 at an MOI of 0.1 for 48 h. Data are expressed as mean ± SD. n = 6 biological replicates. ns, not significant; ****, p < 0.0001.

Figure 4

The antiviral activity of EIDD-1931, remdesivir, nirmatrelvir, and other small molecules in ACE2plusC3 cells. Cells were infected with WT SARS-CoV-2 at an MOI of 0.1 for 48 h in the presence of the indicated concentrations (μM). Infection was calculated by dividing the number of infected cells (measured by immunofluorescence of SARS-CoV-2 nucleocapsid protein) by the total cell nuclei present (measured by DAPI). The red line indicates the inhibition of SARS-CoV-2 infection, and the blue line indicates cell viability. Each condition was performed in sextuplicate with averages and standard deviations indicated. The curves were fitted using GraphPad Prism software. Half maximal inhibitory concentration (IC₅₀) was calculated from the curve fit. Cell viability was measured by LDH assay and by cell morphology and cell number.

Figure 5

The antiviral activity of EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir against wild-type, delta, and omicron SARS-CoV-2 infection in ACE2plusC3 cells. Small molecules were evaluated in ACE2plusC3 cells at the indicated concentrations (μM) or with DMSO control. Cells were infected with wild-type SARS-CoV-2 and the indicated variants at an MOI of 0.1 for 48 h. Virus-infected cells were visualized with immunofluorescence staining of SARS-CoV-2 NP. Infectivity was measured as described in Figure 1. Each concentration was performed in sextuplicate with averages and standard deviations indicated. ns, not significant; ***, p < 0.001; ****, p < 0.0001.