RESUMEN
Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.
Asunto(s)
Antivirales , Receptores de Esteroides , SARS-CoV-2 , Proteínas no Estructurales Virales , Replicación Viral , Replicación Viral/efectos de los fármacos , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Antivirales/farmacología , Receptores de Esteroides/metabolismo , Proteínas no Estructurales Virales/metabolismo , COVID-19/virología , COVID-19/metabolismo , Chlorocebus aethiops , Células Vero , Proteínas Virales/metabolismo , Células HEK293 , Animales , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteínas Viroporinas/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Unión ProteicaRESUMEN
Rationale: Human coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively. Methods: Primary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication. Results: Both CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model. Conclusion: The immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.