The study investigates the role of non-structural protein 1 (Nsp1) of SARS-CoV-2 in facilitating viral replication through the calcineurin-NFAT signaling pathway. Nsp1 interacts with calcineurin A (CnA) to displace the regulatory protein RCAN3, leading to NFAT activation and the upregulation of cellular DEAD-box helicase 5 (DDX5), which enhances viral replication. The study also demonstrates that calcineurin inhibitors, such as cyclosporine A (CsA) and VIVIT, inhibit SARS-CoV-2 replication and exhibit synergistic antiviral effects when combined with nirmatrelvir, a main protease inhibitor. The findings provide insights into the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors.The study investigates the role of non-structural protein 1 (Nsp1) of SARS-CoV-2 in facilitating viral replication through the calcineurin-NFAT signaling pathway. Nsp1 interacts with calcineurin A (CnA) to displace the regulatory protein RCAN3, leading to NFAT activation and the upregulation of cellular DEAD-box helicase 5 (DDX5), which enhances viral replication. The study also demonstrates that calcineurin inhibitors, such as cyclosporine A (CsA) and VIVIT, inhibit SARS-CoV-2 replication and exhibit synergistic antiviral effects when combined with nirmatrelvir, a main protease inhibitor. The findings provide insights into the molecular mechanism of CsA-mediated inhibition of SARS-CoV-2 replication and the anti-SARS-CoV-2 action of calcineurin inhibitors.