Potential coupling between SARS-CoV-2 replicative fitness and interactions of its nucleoprotein with human 14-3-3 proteins

The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.

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The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

10.1101/2020.12.26.424450 ◽

2020 ◽

Author(s):

Kristina V. Tugaeva ◽

Dorothy E. D. P. Hawkins ◽

Jake L. R. Smith ◽

Oliver W. Bayfield ◽

De-Sheng Ker ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Phosphorylation Site ◽

Dependent Manner ◽

Nucleocytoplasmic Shuttling ◽

Genome Packaging ◽

Rich Region ◽

N Protein ◽

Proteomic Data ◽

Cellular Pathways ◽

Micromolar Range

AbstractThe coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Phosphorylated SARS-CoV N was shown to bind to the host 14-3-3 protein in the cytoplasm. Proteomic data indicate that seven human 14-3-3 proteins are highly abundant in human tissues vulnerable to SARS-CoV-2 infection, collectively reaching ~1.8% of all proteins in the lungs, ~1.4% in the gastrointestinal system, ~2.3% in the nervous system. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its mechanism and the specific critical phosphosites were 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 hub, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and determine the apparent KD to selected isoforms in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, located within the SR-rich region of N. The tight 14-3-3/pN association suggests it could regulate nucleocytoplasmic shuttling of N, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention.

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The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

10.1101/2021.12.25.474155 ◽

2021 ◽

Author(s):

Christen E Tai ◽

Einav Tayeb-Fligelman ◽

Sarah Griner ◽

Lukasz Salwinski ◽

Jeannette T Bowler ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Amyloid Fibrils ◽

Rna Binding ◽

Low Complexity ◽

Dimerization Domain ◽

Mobility Shift ◽

Genome Packaging ◽

Biochemical Basis ◽

Virion Assembly ◽

Multiple Structures

The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.

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Structural basis for proficient oxidized ribonucleotide insertion in double strand break repair

Nature Communications ◽

10.1038/s41467-021-24486-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joonas A. Jamsen ◽

Akira Sassa ◽

Lalith Perera ◽

David D. Shock ◽

William A. Beard ◽

...

Keyword(s):

Mammalian Cells ◽

Time Lapse ◽

Strand Break ◽

Structural Basis ◽

End Joining ◽

Strand Breaks ◽

Nucleotide Pools ◽

Nucleotide Insertion ◽

Non Homologous End Joining

AbstractReactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability.

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The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

Nature Communications ◽

10.1038/s41467-020-20768-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shan Lu ◽

Qiaozhen Ye ◽

Digvijay Singh ◽

Yong Cao ◽

Jolene K. Diedrich ◽

...

Keyword(s):

Phase Separation ◽

Potential Role ◽

Rna Binding ◽

Stress Granule ◽

M Protein ◽

Rich Region ◽

Virion Assembly ◽

N Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

AbstractThe multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80–90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein’s central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

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Structural basis for a complex I mutation that blocks pathological ROS production

Nature Communications ◽

10.1038/s41467-021-20942-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhan Yin ◽

Nils Burger ◽

Duvaraka Kula-Alwar ◽

Dunja Aksentijević ◽

Hannah R. Bridges ◽

...

Keyword(s):

Point Mutation ◽

Complex I ◽

Structural Changes ◽

Ischemia Reperfusion ◽

Single Point ◽

Structural Basis ◽

Single Point Mutation ◽

Ros Production

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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Crystal structure of human carbonic anhydrase II at 1.95 Å resolution in complex with 667-coumate, a novel anti-cancer agent

Biochemical Journal ◽

10.1042/bj20041037 ◽

2005 ◽

Vol 385 (3) ◽

pp. 715-720 ◽

Cited By ~ 32

Author(s):

Matthew D. LLOYD ◽

Richard L. PEDERICK ◽

Ramanathan NATESH ◽

L. W. Lawrence WOO ◽

Atul PUROHIT ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Phase 1 ◽

Structural Basis ◽

Pharmacokinetic Studies ◽

Reversible Binding ◽

X Ray Crystallography ◽

Hydrophobic Binding ◽

Cancer Agent

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-Coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 Å (1 Å=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 Å through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

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Methods to study the structure of misfolded protein states in systemic amyloidosis

Biochemical Society Transactions ◽

10.1042/bst20201022 ◽

2021 ◽

Vol 49 (2) ◽

pp. 977-985

Author(s):

Marcus Fändrich ◽

Matthias Schmidt

Keyword(s):

Protein Misfolding ◽

Amyloid Fibrils ◽

Ex Vivo ◽

Systemic Amyloidosis ◽

Structural Basis ◽

Amyloid A ◽

Serum Amyloid A Protein ◽

Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

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Cotranslational Coat Protein-Mediated Inhibition of Potyviral RNA Translation

Journal of Virology ◽

10.1128/jvi.02915-14 ◽

2015 ◽

Vol 89 (8) ◽

pp. 4237-4248 ◽

Cited By ~ 22

Author(s):

Jane Besong-Ndika ◽

Konstantin I. Ivanov ◽

Anders Hafrèn ◽

Thierry Michon ◽

Kristiina Mäkinen

Keyword(s):

Coat Protein ◽

Stop Codon ◽

Rna Virus ◽

Viral Rna ◽

Dependent Manner ◽

Content Type ◽

Virion Assembly ◽

Rna Translation ◽

Intrinsic Capacity

ABSTRACTPotato virus A(PVA) is a single-stranded positive-sense RNA virus and a member of the familyPotyviridae. The PVA coat protein (CP) has an intrinsic capacity to self-assemble into filamentous virus-like particles, but the mechanism responsible for the initiation of viral RNA encapsidationin vivoremains unclear. Apart from virion assembly, PVA CP is also involved in the inhibition of viral RNA translation. In this study, we show that CP inhibits PVA RNA translation in a dose-dependent manner, through a mechanism involving the CP-encoding region. Analysis of this region, however, failed to identify any RNA secondary structure(s) preferentially recognized by CP, suggesting that the inhibition depends on CP-CP rather than CP-RNA interactions. In agreement with this possibility, insertion of an in-frame stop codon upstream of the CP sequence led to a marked decrease in the inhibition of viral RNA translation. Based on these results, we propose a model in which the cotranslational interactions between excess CP accumulating intransand CP translated from viral RNA incisare required to initiate the translational repression. This model suggests a mechanism for how viral RNA can be sequestered from translation and specifically selected for encapsidation at the late stages of viral infection.IMPORTANCEThe main functions of the CP during potyvirus infection are to protect viral RNA from degradation and to transport it locally, systemically, and from host to host. Although virion assembly is a key step in the potyviral infectious cycle, little is known about how it is initiated and how viral RNA is selected for encapsidation. The results presented here suggest that CP-CP rather than CP-RNA interactions are predominantly involved in the sequestration of viral RNA away from translation. We propose that the cotranslational nature of these interactions may represent a mechanism for the selection of viral RNA for encapsidation. A better understanding of the mechanism of virion assembly may lead to development of crops resistant to potyviruses at the level of viral RNA encapsidation, thereby reducing the detrimental effects of potyvirus infections on food production.

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Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies

Frontiers in Immunology ◽

10.3389/fimmu.2021.719037 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qiaozhen Ye ◽

Shan Lu ◽

Kevin D. Corbett

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Binding Domain ◽

Single Domain ◽

Structural Basis ◽

Dimerization Domain ◽

Health Event ◽

Single Domain Antibodies ◽

Rna Binding Domain ◽

Antigen Tests

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

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Mechanosensation and Mechanotransduction in Natural Killer Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.688918 ◽

2021 ◽

Vol 12 ◽

Author(s):

Giorgio Santoni ◽

Consuelo Amantini ◽

Matteo Santoni ◽

Federica Maggi ◽

Maria Beatrice Morelli ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Target Cell ◽

Nk Cell ◽

Tyrosine Phosphatase ◽

Cell Interaction ◽

Structural Basis ◽

Adhesive Interactions

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

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