RESUMEN
Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO4 3-) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano- LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2-infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Polifosfatos/farmacología , SARS-CoV-2/efectos de los fármacos , Administración por Inhalación , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Antivirales/administración & dosificación , Antivirales/química , COVID-19/metabolismo , COVID-19/virología , Células CACO-2 , Chlorocebus aethiops , ARN Polimerasa Dependiente de ARN de Coronavirus/química , ARN Polimerasa Dependiente de ARN de Coronavirus/genética , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Citocinas/metabolismo , Células HEK293 , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/fisiología , Humanos , Técnicas In Vitro , Modelos Biológicos , Simulación del Acoplamiento Molecular , Nebulizadores y Vaporizadores , Polifosfatos/administración & dosificación , Polifosfatos/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteolisis/efectos de los fármacos , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Progression through mitosis, the cell cycle phase deputed to segregate replicated chromosomes, is granted by a protein phosphorylation wave that follows an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1, the major mitosis-promoting enzyme. To ensure correct chromosome segregation, the safeguard mechanism spindle assembly checkpoint (SAC) delays Cdk1 inactivation by preventing cyclin B degradation until mitotic spindle assembly. At the end of mitosis, reversal of bulk mitotic protein phosphorylation, downstream Cdk1 inactivation, is required to complete mitosis and crucially relies on the activity of major protein phosphatases like PP2A. A role for PP2A, however, has also been suggested in spindle assembly and SAC-dependent control of Cdk1. Indeed, PP2A was found in complex with SAC proteins while small interfering RNAs (siRNAs)-mediated downregulation of PP2A holoenzyme components affected mitosis completion in mammalian cells. However, whether the SAC-dependent control of Cdk1 required the catalytic activity of PP2A has never been directly assessed. Here, using two PP2A inhibitors, okadaic acid and LB-100, we provide evidence that PP2A activity is dispensable for SAC control of Cdk1 in human cells.