Scientists are developing human cell lines suitable for anti-SARS-CoV-2 drug screening

In an article recently published in the journal virusScientists describe the development of a human cell line suitable for high-throughput screening of antiviral drugs targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Their scientists have found high levels of angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), Two important host proteins required for viral entry.

Background

The 2019 coronavirus disease (COVID-19) pandemic caused by SARS-CoV-2 has placed an immeasurable burden on the global medical system, with more than 359 million COVID-19 cases identified. , More than 6.3 million people have died worldwide. As the pandemic progresses, many viral variants with new mutational landscapes are emerging worldwide. Some of them have been designated as Variant of Concern (VOCs) due to their significant improvements in transmissibility, infectivity, pathogenicity, and antigenic escape. Given the growing pandemic threat to global public health, there is an urgent need to identify and develop SARS-CoV-2 specific antivirals.

In the current study, scientists describe the development of a modified A549 cell line that exhibits strong expression of ACE2 and TMPRSS2 and high susceptibility to SARS-CoV-2 infection. ACE2 and TMPRSS2 are essential for hosting the proteins required for SARS-CoV-2 to invade host cells.

Modification of A549 cell line

Modification of the A549 cell line was performed by transducing human ACE2-expressing lentivirus into cells and then selecting puromycin-based clones. The selected clones were then transduced with TMPRSS2-expressing lentivirus.

Over 50 clones have been selected. One of these clones showed strong expression of ACE2 and TMPRSS2 and a high SARS-CoV-2 infection rate (60%). The infection rate of modified cell lines is similar to that of the Vero E6 cell line, which is an established and widely used cell line to characterize SARS-CoV-2 infection.

Single cell sorting of these clones was performed to generate subclones derived from a single cell. The susceptibility of these subclones (modified cells) to viral infection was determined by infection with wild-type SARS-CoV-2 and its variants Delta and Omicron. Overall, the modified cells showed high susceptibility to infection by all the viral variants tested. The infection rate of modified cells was higher than that of commercially available A549 cell lines expressing human ACE2 and TMPRSS2.

Characteristics of modified A549 cell line

Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were performed to measure the mRNA expression of ACE2 and TMPRSS2 and the cell surface expression of ACE2, respectively.

The findings revealed that the modified cells express both ACE2 and TMPRSS2 at significantly higher levels than the commercially available A549 cells that express ACE2 and TMPRSS2. In addition, the modified cells showed higher infectivity to SARS-CoV-2 containing the D614G mutation compared to wild-type virus. D614G is known to block ACE2 receptor binding and viral entry.

Application of modified A549 cells

The usefulness of modified A549 cells as an antiviral screening platform was tested in this study.Specifically, we compared antiviral agents using modified cells infected with SARS-CoV-2. Effectiveness of Nine antiviral drugs (recycled anti-COVID 19 drug nilmatrelvir, EIDD-1931, remdecibir, anti-HIV drug nelfinavir, and widely used antiviral candidate camostat mesylate, naphthofluoresane, E64d, and Decanoyl-RVKR-CMK).

Dose-response analysis revealed that all reused and anti-HIV drugs strongly inhibit SARS-CoV-2 infection in modified cells. Of the candidate drugs, only camostat mesylate significantly suppressed viral infection. These observations show that the modified cells can be used for high-throughput screening of antiviral drugs.

In addition, modified cells were used to test the antiviral effect of the panel of drugs against delta and omicron infections. All antiviral drugs tested (EIDD-1931, remdesivir, nilmatrelvir, nelfinavir) showed dose-dependent inhibition of infections caused by wild-type SARS-CoV-2 and delta and omicron mutants. .. In the modified cells, the Omicron mutant showed higher sensitivity to drug treatment compared to the wild-type SARS-CoV-2 and Delta mutants.

Significance of research

In this study, a human cell model suitable for SARS-CoV-2 infection was developed. Lentivirus transduction is used for stable and robust expression of human ACE2 and TMPRSS2 in A549 cells. As scientists suggest, these modified A549 cells could be used to study new SARS-CoV-2 mutants and to screen for antiviral drugs.