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CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

ABSTRACT: The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

SUBMITTER: Verba K

PROVIDER: S-EPMC8142659 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants.ResultsHere, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface.ConclusionOur study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Project description:BackgroundPeriventricular extracellular oedema, myelin damage, inflammation, and glial reactions are common neuropathological events that occur in the brain in congenital hydrocephalus. The periventricular white matter is the most affected region. The present study aimed to identify altered molecular and cellular biomarkers in the neocortex that can function as potential therapeutic targets to both treat and evaluate recovery from these neurodegenerative conditions. The hyh mouse model of hereditary hydrocephalus was used for this purpose.MethodsThe hyh mouse model of hereditary hydrocephalus (hydrocephalus with hop gait) and control littermates without hydrocephalus were used in the present work. In tissue sections, the ionic content was investigated using energy dispersive X-ray spectroscopy scanning electron microscopy (EDS-SEM). For the lipid analysis, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was performed in frozen sections. The expression of proteins in the cerebral white matter was analysed by mass spectrometry. The oligodendrocyte progenitor cells (OPCs) were studied with immunofluorescence in cerebral sections and whole-mount preparations of the ventricle walls.ResultsHigh sodium and chloride concentrations were found indicating oedema conditions in both the periventricular white matter and extending towards the grey matter. Lipid analysis revealed lower levels of two phosphatidylinositol molecular species in the grey matter, indicating that neural functions were altered in the hydrocephalic mice. In addition, the expression of proteins in the cerebral white matter revealed evident deregulation of the processes of oligodendrocyte differentiation and myelination. Because of the changes in oligodendrocyte differentiation in the white matter, OPCs were also studied. In hydrocephalic mice, OPCs were found to be reactive, overexpressing the NG2 antigen but not giving rise to an increase in mature oligodendrocytes. The higher levels of the NG2 antigen, diacylglycerophosphoserine and possibly transthyretin in the cerebrum of hydrocephalic hyh mice could indicate cell reactions that may have been triggered by inflammation, neurocytotoxic conditions, and ischaemia.ConclusionOur results identify possible biomarkers of hydrocephalus in the cerebral grey and white matter. In the white matter, OPCs could be reacting to acquire a neuroprotective role or as a delay in the oligodendrocyte maturation.

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CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

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