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

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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Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

10.1101/2021.06.14.448452 ◽

2021 ◽

Author(s):

Christine Roden ◽

Yifan Dai ◽

Ian Seim ◽

Myungwoon Lee ◽

Rachel Sealfon ◽

...

Keyword(s):

Phase Separation ◽

Rna Structure ◽

Nucleocapsid Protein ◽

Rna Binding ◽

Genome Packaging ◽

Rna Motifs ◽

Double Stranded Rna ◽

N Protein ◽

Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

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Targeting the coronavirus nucleocapsid protein through GSK-3 inhibition

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2113401118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2113401118

Author(s):

Xiaolei Liu ◽

Anurag Verma ◽

Gustavo Garcia ◽

Holly Ramage ◽

Anastasia Lucas ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Glycogen Synthase ◽

Lung Epithelial Cells ◽

Dependent Manner ◽

Virion Assembly ◽

N Protein ◽

Consensus Sequences ◽

Lung Epithelial ◽

A Cell ◽

Transcription And Replication

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35–0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type–dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

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Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition

10.1101/2021.02.17.21251933 ◽

2021 ◽

Cited By ~ 1

Author(s):

Xiaolei Liu ◽

Anurag Verma ◽

Holly Ramage ◽

Gustavo Garcia ◽

Rebecca L. Myers ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Glycogen Synthase ◽

Lung Epithelial Cells ◽

Dependent Manner ◽

Virion Assembly ◽

N Protein ◽

Consensus Sequences ◽

Lung Epithelial ◽

A Cell ◽

Transcription And Replication

AbstractThe coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, despite limited overall sequence conservation, raising the possibility that SARS- CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID- 19 (odds ratio = 0.51 [0.34 - 0.76], p = 0.001). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner.Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks.

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SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans

10.1101/2020.08.26.269043 ◽

2020 ◽

Cited By ~ 1

Author(s):

Nitin T. Supekar ◽

Asif Shajahan ◽

Anne S. Gleinich ◽

Daniel Rouhani ◽

Christian Heiss ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Vaccine Development ◽

Point Of Care ◽

Variable Mass ◽

Phosphorylation Site ◽

Post Translational Modifications ◽

N Protein ◽

Point Of Care Diagnostics ◽

Glycosylation Sites

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

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

10.1101/2021.12.23.474009 ◽

2021 ◽

Author(s):

Kristina V Tugaeva ◽

Andrey A. Sysoev ◽

Jake L. R. Smith ◽

Richard B Cooley ◽

Alfred A. Antson ◽

...

Keyword(s):

Fluorescence Anisotropy ◽

Nucleocapsid Protein ◽

Structural Basis ◽

Genome Packaging ◽

Rich Region ◽

Virion Assembly ◽

Replicative Fitness ◽

Multisite Phosphorylation ◽

Human 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 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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SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽

10.3390/cells10030530 ◽

2021 ◽

Vol 10 (3) ◽

pp. 530

Author(s):

Soo Jin Oh ◽

Ok Sarah Shin

Keyword(s):

Nucleocapsid Protein ◽

Nuclear Translocation ◽

Nonstructural Protein ◽

Poly I:C ◽

Interferon Response ◽

Type I ◽

Antiviral Immune Response ◽

N Protein ◽

Nonstructural Protein 1 ◽

Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

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Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

Journal of Virology ◽

10.1128/jvi.78.15.8281-8288.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8281-8288 ◽

Cited By ~ 53

Author(s):

M. A. Mir ◽

A. T. Panganiban

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Rna Viruses ◽

Sin Nombre Virus ◽

Viral Capsid ◽

Genomic Rna ◽

Rna Molecules ◽

N Protein ◽

High Affinity ◽

Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

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The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

PLoS Pathogens ◽

10.1371/journal.ppat.1009931 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009931

Author(s):

Jorge Vera-Otarola ◽

Estefania Castillo-Vargas ◽

Jenniffer Angulo ◽

Francisco M. Barriga ◽

Eduard Batlle ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Initiation Factor ◽

Cellular Proteins ◽

Dependent Manner ◽

The Andes ◽

Viral Nucleocapsid

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

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EGR1 suppresses porcine epidemic diarrhea virus replication by regulating IRAV to degrade viral nucleocapsid protein

Journal of Virology ◽

10.1128/jvi.00645-21 ◽

2021 ◽

Author(s):

Hua Wang ◽

Ning Kong ◽

Yajuan Jiao ◽

Sujie Dong ◽

Dage Sun ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Nucleocapsid Protein ◽

E3 Ubiquitin Ligase ◽

Economic Losses ◽

Restriction Factor ◽

N Protein ◽

Host Restriction ◽

Diarrhea Virus ◽

Protein Ubiquitination ◽

Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus that has re-emerged lately, resulting in large economic losses. During viral infection, interferon (IFN-I) plays a vital role in the antiviral innate immunity. However, PEDV has evolved strategies to limit IFN-I production. To suppress virus replication, the host must activate the IFN-stimulated genes and some host restriction factors to circumvent viral replication. This study observed that PEDV infection-induced early growth response gene 1 (EGR1) expression in PEDV-permissive cells. EGR1 overexpression remarkably suppressed PEDV replication. In contrast, depletion of EGR1 led to a significant increase in viral replication. EGR1 suppressed PEDV replication by directly binding to the IFN-regulated antiviral (IRAV) promoter and upregulating IRAV expression. A detailed analysis revealed that IRAV interacts and colocalizes with the PEDV nucleocapsid (N) protein, inducing N protein degradation via E3 ubiquitin ligase MARCH8 to catalyze N protein ubiquitination. Knockdown of endogenous MARCH8 significantly reversed IRAV-mediated N protein degradation. The collective findings demonstrate a new mechanism of EGR1-mediated viral restriction, in which EGR1 upregulates the expression of IRAV to degrade PEDV N protein through MARCH8. IMPORTANCE PEDV is a highly contagious enteric coronavirus that has rapidly emerged worldwide and caused severe economic losses. No currently available drugs or vaccines could effectively control PEDV. PEDV has evolved many strategies to limit IFN-1 production. We identified EGR1 as a novel host restriction factor and demonstrated that EGR1 suppresses PEDV replication by directly binding to the IRAV promoter and upregulating the expression of IRAV, which interacts and degrades the PEDV N protein via E3 ubiquitin ligase MARCH8 to catalyze nucleocapsid protein ubiquitination, which adds another layer of complexity to innate antiviral immunity of this newly identified restriction factor. A better understanding of the innate immune response to PEDV infection will aid the development of novel therapeutic targets and more effective vaccines against virus infection.

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