Abstract
Background
COVID-19 is a pandemic disease caused by the novel coronavirus SARS-CoV-2, which spread worldwide, revealing uphill repercussions. The infection is mediated by coronavirus spike protein and host cell receptor ACE2 (Angiotensin Converting Enzyme 2). Several cell lines have been used as an in vitro model to test potential drugs and to study the viral kinetics. Since lungs are one of the first organs affected by the virus, pulmonary cell lines such as: A549 (lung cells) and BEAS-2B (Bronchial epithelium cells) are the most studied. However, these cells present a slow viral replication profile which complicate the testing of new antiviral drugs and viral replication studies. High levels of ACE2, which is observed in some groups of patients, is associated with the increase of viral replication and severe symptoms. Thus, the development of a BEAS-2B cell line overexpressing ACE2 would be a useful model to study SARS-CoV-2 infection and to study new drugs.
Aims
Develop a BEAS-2B cell line overexpressing ACE2 and evaluate the role of ACE2 on SARS-CoV-2 viral kinetics.
Methods
BEAS-2B were transfected with pCEP-ACE2-myc and selected with hygromycin (125ng/ul) for 20 days, creating BEAS-2B-ACE2 cell line. ACE2 expression was quantified by RT-qPCR. Western Blotting and Immunofluorescence were used to quantify the ACE2 protein levels. ACE2 activity was evaluated using (MCA-Ala-Pro-Lys(Dnp)-OH) substrate. Cells were infected with SARS-CoV-2 (MOI=0.2). Viral kinetics were analyzed by RT-qPCR. Proliferation analysis was performed by MTT assay.
Results
Long-term overexpression ACE2 in BEAS-2B-ACE2 was confirmed by RT-qPCR, Western Blotting and Immunofluorescence. Compared to BEAS-2B, ACE2 mRNA expression was 100-fold higher (p<0.05), ACE2 protein levels increased 12X (p<0.05) and the immunofluorescence showed that ACE2 protein was more abundantly in BEAS-2B-ACE2. A 50X (p<0.0001) increase in the ACE2 activity was observed 2 months after the transfection. There was no difference in proliferation between BEAS-2B-ACE2 and BEAS-2B. Overexpression of ACE2 increased the viral kinetics. BEAS-2B-ACE2 presented 1000X (p<0.05) more SARS-CoV-2 RNA in cells and supernatant compared with BEAS-2B, 48 and 72 hours after infection.
Conclusion
Here we show for the first time that overexpression of ACE2 in BEAS-2B drastically changes the infection profile of SARS-CoV-2 and increases viral load – making it an useful cell line for future studies of SARS-CoV-2.