The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ∼1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent K D to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.
The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.HighlightsSARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.
We have reported that CD-6′SLN [6-sialyllactosamine (6′SLN)-modified β-cyclodextrin (CD)] can be a potential anti-influenza drug because it irreversibly deactivates virions. Indeed, in vivo, CD-6′SLN improved mice survival in an H1N1 infection model even when administered 24 h post-infection. Although CD-6′SLN was designed to target the viral envelope protein hemagglutinin (HA), a natural receptor of 6′SLN, it remains unclear whether other targets exist. In this study, we confirm that CD-6′SLN inhibits the influenza virus through an extracellular mechanism by interacting with HA, but not with neuraminidase (NA), despite the latter also having a binding pocket for the sialyl group. We find that CD-6′SLN interacts with the viral envelope as it elicits the release of a fluorophore embedded in the membrane. Two similar compounds were designed to test separately the effect of 6′SLN and of the undecyl moiety that links the CD to 6′SLN. Neither showed any interaction with the membrane nor the irreversible viral inhibition (virucidal), confirming that both components are essential to membrane interaction and virucidal action. Unlike similar antiviral cyclodextrins developed against other viruses, CD-6′SLN was not able to decapsulate viral RNA. Our findings support that combining viral protein-specific epitopes with hydrophobic linkers provides a strategy for developing antiviral drugs with a virucidal mechanism.
Испытаны составы пеногелей, структурированные нанодобавками и добавками, бронирующими пленку. В качестве электролитов коагуляторов, т. е. веществ, используемых для гелеобразования, применяют соли сильных кислот или сами кислоты. Полученные в результате составы пеногелей характеризуются примерно одинаковыми свойствами по кратности и устойчивости во времени, что отвечает требованиям их использования при проведении взрывных работ. Анализ составов, полученных с использованием хлорида железа, медного купороса и хлорида бария, показал, что наиболее стабильный состав получается с хлоридом железа и ортофосфорной кислотой. В качестве нанодобавки наиболее приемлемым вариантом признан «наносил-30». Пеногель заполнялся в ампулы, которые используются при изготовлении забойки шпуров.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.