In an article recently published in the journal Viruses, scientists have described the development of a suitable human cell line for high-throughput screening of antiviral drugs targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
They scientists engineered human lung carcinoma cell line A549 to express high levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), two vital host proteins required for viral entry.
The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has put an immense burden on the global healthcare system, with more than 539 million confirmed COVID-19 cases and over 6.3 million deaths worldwide. With the progression of the pandemic, many viral variants with novel mutational landscapes have emerged globally. Some of them have been designated as the variants of concern (VOCs) because of significantly improved transmissibility, infectivity, virulence, and immune evasion. Given the increasing threat of the pandemic on global public health, identification and development of SARS-CoV-2 specific antiviral drugs are urgently needed.
In the current study, the scientists have described the development of a modified A549 cell line that shows a robust expression of ACE2 and TMPRSS2 and high susceptibility to SARS-CoV-2 infection. ACE2 and TMPRSS2 are vital to host proteins that are required for SARS-CoV-2 entry into host cells.
Modification of A549 cell line
The modification of A549 cell line was done by transducing the cells with human ACE2-expressing lentivirus, followed by puromycin-based selection of clones. Afterward, selected clones were transduced with TMPRSS2-expressing lentivirus.
More than 50 clones were selected. Of which, one clone showed robust ACE2 and TMPRSS2 expression and a high SARS-CoV-2 infection rate (60%). The infection rate in the modified cell line is similar to that in the Vero E6 cell line, which is a well-established and widely used cell line for characterizing SARS-CoV-2 infection.
Single-cell sorting of these clones was conducted to generate single-cell-derived subclones. Susceptibility of these subclones (modified cells) to viral infection was determined by infecting them with wildtype SARS-CoV-2 and its variants delta and omicron. Overall, the modified cells showed high susceptibility to infection by all tested viral variants. The infection rate of the modified cells was higher than a commercially available human ACE2- and TMPRSS2-expressing A549 cell line.
Characterization of modified A549 cell line
Reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were conducted to determine the mRNA expressions of ACE2 and TMPRSS2 and cell surface expression of ACE2, respectively.
The findings revealed that the modified cells express significantly higher levels of both ACE2 and TMPRSS2 than the commercially available ACE2- and TMPRSS2-expressing A549 cells. Moreover, the modified cells showed higher infectivity for SARS-CoV-2 containing D614G mutation compared to the wild-type virus. The D614G is known to interfere with ACE2 receptor binding and viral entry.
Application of modified A549 cells
The utility of modified A549 cells as a screening platform for antiviral drugs was tested in the study. Specifically, SARS-CoV-2-infected modified cells were used to compare the antiviral efficacy of nine antiviral drugs (repurposed anti-COVID 19 drugs Nirmatrelvir, EIDD-1931, remdesivir; anti-HIV drug Nelfinavir; and widely-used antiviral drug candidates camostat mesylate, naphthofluorescein, E64d, and decanoyl-RVKR-CMK).
The dose-response analysis revealed that all repurposed drugs as well as the anti-HIV drug strongly inhibit SARS-CoV-2 infection in modified cells. Among drug candidates, only camostat mesylate significantly inhibited the viral infection. These observations indicate that the modified cells can be used for high-throughput screening of antiviral drugs.
Furthermore, the modified cells were used to test the antiviral efficacy of a panel of drugs against delta and omicron infections. All tested antiviral drugs (EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir) showed dose-dependent inhibition of infection caused by wildtype SARS-CoV-2 and delta and omicron variants. In the modified cells, the omicron variant showed higher sensitivity to the drug treatment compared to wildtype SARS-CoV-2 and the delta variant.
A human cell model suitable for SARS-CoV-2 infection has been developed in the study. Lentiviral transduction method has been used to stably and robustly express human ACE2 and TMPRSS2 in A549 cells. As suggested by the scientists, these modified A549 cells could be potentially used to study emerging SARS-CoV-2 variants and screen antiviral drugs.