ABSTRACT
The most common host entry point of human adapted coronaviruses (CoV) including SARS-CoV-2 is through the initial colonization in the nostril and mouth region which is responsible for spread of the infection. Most recent studies suggest that the commercially available oral and nasal rinse products are effective in inhibiting the viral replication. However, the anti-viral mechanism of the active ingredients present in the oral rinses have not been studied. In the present study, we have assessed in vitro enzymatic inhibitory activity of active ingredients in the oral mouth rinse products: aloin A and B, chlorhexidine, eucalyptol, hexetidine, menthol, triclosan, methyl salicylate, sodium fluoride and povidone, against two important proteases of SARS-CoV-2 PLpro and 3CLpro. Our results indicate only aloin A and B effectively inhibited proteolytic activity of PLpro with an IC50 of 13.16 and 16.08 µM. Interestingly, neither of the aloin isoforms inhibited 3CLpro enzymatic activity. Computational structural modelling of aloin A and B interaction with PLpro revealed that, both aloin isoforms form hydrogen bond with Tyr268 of PLpro, which is critical for their proteolytic activity. Furthermore, 100 ns molecular dynamics (MD) simulation studies predicted that both aloin isoforms have strong interaction with Glu167, which is required for PLpro deubiquitination activity. Our results from the in vitro deubiquitinase inhibition assay show that aloin A and B isomers exhibit deubiquitination inhibitory activity with an IC50 value of 15.68 and 17.51 µM, respectively. In conclusion, the isoforms of aloin inhibit both proteolytic and the deubiquitinating activity of SARS-CoV-2 PLpro, suggesting potential in inhibiting the replication of SARS-CoV-2 virus.
Subject(s)
Coronavirus Papain-Like Proteases/metabolism , Emodin/analogs & derivatives , SARS-CoV-2/enzymology , Animals , Binding Sites , COVID-19/pathology , COVID-19/virology , Cell Survival/drug effects , Chlorocebus aethiops , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Emodin/chemistry , Emodin/metabolism , Emodin/pharmacology , Humans , Molecular Dynamics Simulation , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Isoforms/pharmacology , SARS-CoV-2/isolation & purification , Vero CellsABSTRACT
ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Bile Ducts/metabolism , Bile Ducts/virology , Binding Sites , COVID-19/pathology , COVID-19/virology , Humans , Lung/metabolism , Lung/virology , Microscopy, Fluorescence, Multiphoton , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Virus/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Virus InternalizationABSTRACT
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 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.
Subject(s)
14-3-3 Proteins/chemistry , 14-3-3 Proteins/metabolism , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Amino Acid Sequence , Binding Sites/genetics , Coronavirus Nucleocapsid Proteins/genetics , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Escherichia coli , Humans , Mutation , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Phosphoserine/metabolism , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , RNA, Viral/metabolism , Substrate SpecificityABSTRACT
SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.