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.

Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery

“…For both compounds, the CC 50 was determined to be > 50 μM, resulting in a selectivity index (SI) of >30 for GS-441524 and > 115 for PF-00835231. These values were similar to those that have been reported in the literature ( Abdelnabi et al, 2022 ), ( Chang et al, 2022 ). In total, 32 × 384-well plates were used to confirm the robustness of this assay.…”

“…We evaluated in this assay the antiviral activity against SARS-CoV-2 replication of a selection of SARS-CoV-2 reference inhibitors. The results obtained were concordant to reports from other publications ( Agostini et al, 2019 )– ( Vangeel et al, 2022 ), ( Chang et al, 2022 ), ( Patten et al, 2022 ), ( Softic et al, 2020 ). An additional benefit of our assay is that it allows simultaneous read-out of both the antiviral potential and cytotoxicity caused by the compound.…”

Omicron Subvariants: Clinical, Laboratory, and Cell Culture Characterization

“…When we compared the recovery of infectious virus from different Omicron subvariants on VT versus VAT, we noticed an increased sensitivity with VAT that was more notable for samples with lower relative viral loads. Interestingly, an overexpression of ACE-2 was advantageous for not only Omicron subvariants, but also to the original SARS-CoV-2 and pre-Omicron variants as shown by our TCID 50 comparison and consistent with prior reports (27). Further characterization of these two Omicron subvariants on cell culture might reveal the genomic basis of the increased BA.5 viral fitness noted in our study.…”

Choosing a cellular model to study SARS-CoV-2

“…The A549 cells overexpressing ACE2 has been shown to be valuable tools in anti-SARS-Cov-2 drug screening (Plaze et al, 2021;Pyrcé t al., 2021;Napolitano et al, 2022). A new report of transduced A549 subclones selected to express more ACE2, and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2 highlights its increased susceptibility, including to the Delta and Omicron variants, and its potential as a drug-inhibition cellular model (Chang et al, 2022). Commercial HeLa-bases (derived from cervical cancer cells (Scherer et al, 1953)) and HEK 293T-based cell lines that are genetically modified to express ACE2, TMPRSS2, or both are currently available (Figure 3) (Vectorbuilder).…”