scholarly journals A Comparative Analysis of Coronavirus Nucleocapsid (N) Proteins Reveals the SADS-CoV N Protein Antagonizes IFN-β Production by Inducing Ubiquitination of RIG-I

2021 ◽  
Vol 12 ◽  
Author(s):  
Yan Liu ◽  
Qi-Zhang Liang ◽  
Wan Lu ◽  
Yong-Le Yang ◽  
Ruiai Chen ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Rna Binding ◽  
Binding Activity ◽  
N Protein ◽  
Infectious Bronchitis ◽  
Diarrhea Virus ◽  
Multiple Strategies ◽  
Infected Cells ◽  
Global Threat ◽  
N Proteins

Coronaviruses (CoVs) are a known global threat, and most recently the ongoing COVID-19 pandemic has claimed more than 2 million human lives. Delays and interference with IFN responses are closely associated with the severity of disease caused by CoV infection. As the most abundant viral protein in infected cells just after the entry step, the CoV nucleocapsid (N) protein likely plays a key role in IFN interruption. We have conducted a comprehensive comparative analysis and report herein that the N proteins of representative human and animal CoVs from four different genera [swine acute diarrhea syndrome CoV (SADS-CoV), porcine epidemic diarrhea virus (PEDV), severe acute respiratory syndrome CoV (SARS-CoV), SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), infectious bronchitis virus (IBV) and porcine deltacoronavirus (PDCoV)] suppress IFN responses by multiple strategies. In particular, we found that the N protein of SADS-CoV interacted with RIG-I independent of its RNA binding activity, mediating K27-, K48- and K63-linked ubiquitination of RIG-I and its subsequent proteasome-dependent degradation, thus inhibiting the host IFN response. These data provide insight into the interaction between CoVs and host, and offer new clues for the development of therapies against these important viruses.

scholarly journals Measles Virus Assembly within Membrane Rafts

2000 ◽  
Vol 74 (21) ◽  
pp. 9911-9915 ◽  
Author(s):  
Séverine Vincent ◽  
Denis Gerlier ◽  
Serge N. Manié
Keyword(s):  
G Protein ◽  
Measles Virus ◽  
Virus Assembly ◽  
Membrane Rafts ◽  
Virus Envelope ◽  
N Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Triton X ◽  
N Proteins

ABSTRACT During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305–311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

scholarly journals Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

2005 ◽  
Vol 79 (15) ◽  
pp. 10023-10031 ◽  
Author(s):  
Jens Modrof ◽  
Kostas Lymperopoulos ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Nonstructural Protein 2 ◽  
The Matrix ◽  
Infected Cells ◽  
Viral Inclusion

ABSTRACT In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

scholarly journals The RNA-Binding Domain of Bacteriophage P22 N Protein Is Highly Mutable, and a Single Mutation Relaxes Specificity toward λ

2008 ◽  
Vol 190 (23) ◽  
pp. 7699-7708 ◽  
Author(s):  
Alexis I. Cocozaki ◽  
Ingrid R. Ghattas ◽  
Colin A. Smith
Keyword(s):  
Rna Binding ◽  
Binding Domain ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Bacteriophage P22 ◽  
Resonance Model ◽  
N Protein ◽  
Rna Binding Domain ◽  
N Proteins

ABSTRACT Antitermination in bacteriophage P22, a lambdoid phage, uses the arginine-rich domain of the N protein to recognize boxB RNAs in the nut site of two regulated transcripts. Using an antitermination reporter system, we screened libraries in which each nonconserved residue in the RNA-binding domain of P22 N was randomized. Mutants were assayed for the ability to complement N-deficient virus and for antitermination with P22 boxBleft and boxBright reporters. Single amino acid substitutions complementing P22 N− virus were found at 12 of the 13 positions examined. We found evidence for defined structural roles for seven nonconserved residues, which was generally compatible with the nuclear magnetic resonance model. Interestingly, a histidine can be replaced by any other aromatic residue, although no planar partner is obvious. Few single substitutions showed bias between boxBleft and boxBright, suggesting that the two RNAs impose similar constraints on genetic drift. A separate library comprising only hybrids of the RNA-binding domains of P22, λ, and φ21 N proteins produced mutants that displayed bias. P22 N− plaque size plotted against boxBleft and boxBright reporter activities suggests that lytic viral fitness depends on balanced antitermination. A few N proteins were able to complement both λ N- and P22 N-deficient viruses, but no proteins were found to complement both P22 N- and φ21 N-deficient viruses. A single tryptophan substitution allowed P22 N to complement both P22 and λ N−. The existence of relaxed-specificity mutants suggests that conformational plasticity provides evolutionary transitions between distinct modes of RNA-protein recognition.

scholarly journals Epstein–Barr virus nuclear antigen 1 is a DNA-binding protein with strong RNA-binding activity

2004 ◽  
Vol 85 (10) ◽  
pp. 2755-2765 ◽  
Author(s):  
Chih-Chung Lu ◽  
Chia-Wei Wu ◽  
Shin C. Chang ◽  
Tzu-Yi Chen ◽  
Chwan-Ren Hu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Rna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Binding Activity ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 5′ non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 5′ NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA.

scholarly journals Gammacoronavirus Avian Infectious Bronchitis Virus and Alphacoronavirus Porcine Epidemic Diarrhea Virus Exploit a Cell-Survival Strategy via Upregulation of cFOS to Promote Viral Replication

2020 ◽  
Author(s):  
Li Xia Yuan ◽  
Jia Qi Liang ◽  
Qing Chun Zhu ◽  
Guo Dai ◽  
Shumin Li ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Viral Replication ◽  
Infectious Bronchitis ◽  
Diarrhea Virus ◽  
Intermediate Phases ◽  
A Cell ◽  
Infected Cells ◽  
Coronavirus Infection ◽  
Porcine Epidemic Diarrhea ◽  
Induced Apoptosis

Coronaviruses have evolved a variety of strategies to optimize cellular microenvironment for efficient replication. In this study, we report the induction of AP-1 transcription factors by coronavirus infection based on genome-wide analyses of differentially expressed genes in cells infected with avian coronavirus infectious bronchitis virus (IBV). Most members of the AP-1 transcription factors were subsequently found to be upregulated during the course of IBV and porcine epidemic diarrhea virus (PEDV) infection of cultured cells as well as in IBV-infected chicken embryos. Further characterization of the induction kinetics and functional roles of cFOS in IBV replication demonstrated that upregulation of cFOS at early to intermediate phases of IBV replication cycles suppresses IBV-induced apoptosis and promotes viral replication. Blockage of nuclear translocation of cFOS by peptide inhibitor NLSP suppressed IBV replication and apoptosis, ruling out the involvement of the cytoplasmic functions of cFOS in the replication of IBV. Furthermore, knockdown of ERK1/2 and inhibition of JNK and p38 kinase activities reduced cFOS upregulation and IBV replication. This study reveals an important function of cFOS in the regulation of coronavirus-induced apoptosis, facilitating viral replication. IMPORTANCE The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by a newly emerged zoonotic coronavirus (SARS-CoV-2), highlights the importance of coronaviruses as human and animal pathogens and our knowledge gaps in understanding the cellular mechanisms, especially mechanisms shared among human and animal coronaviruses, exploited by coronaviruses for optimal replication and enhanced pathogenicity. This study reveals that upregulation of cFOS, along with other AP-1 transcription factors, as a cell-survival strategy is such a mechanism utilized by coronaviruses during their replication cycles. Through induction and regulation of apoptosis of the infected cells at early to intermediate phases of the replication cycles, subtle but appreciable differences in coronavirus replication efficiency were observed when the expression levels of cFOS were manipulated in the infected cells. As the AP-1 transcription factors are multi-functional, further studies of their regulatory roles in proinflammatory responses may provide new insights into the pathogenesis and virus-host interactions during coronavirus infection.

scholarly journals Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance

2005 ◽  
Vol 79 (2) ◽  
pp. 1164-1179 ◽  
Author(s):  
Hongying Chen ◽  
Andrew Gill ◽  
Brian K. Dove ◽  
Stevan R. Emmett ◽  
C. Fred Kemp ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Protein Binding ◽  
Binding Site ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Infectious Bronchitis Virus ◽  
Rna Binding ◽  
Viral Rna ◽  
N Protein ◽  
Infectious Bronchitis

ABSTRACT Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

scholarly journals Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

2021 ◽  
Author(s):  
Helder Veras Ribeiro Filho ◽  
Gabriel Ernesto Jara ◽  
Fernanda Aparecida Heleno Batista ◽  
Gabriel Ravanhani Schleder ◽  
Celisa Caldana Tonoli ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Binding ◽  
Structural Data ◽  
Binding Activity ◽  
Full Length ◽  
N Protein ◽  
Intrinsically Disordered ◽  
Virus Life Cycle ◽  
Biophysical Techniques ◽  
Dynamics Simulations

AbstractThe nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two structured RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein forms structurally dynamic dimers with the NTD and CTD arranged in extended conformations. In the presence of RNA, however, the N protein assumes a more compact conformation where the NTD and CTD are packed together. We also provide an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

scholarly journals Subcellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein

2005 ◽  
Vol 86 (12) ◽  
pp. 3303-3310 ◽  
Author(s):  
Jaehwan You ◽  
Brian K. Dove ◽  
Luis Enjuanes ◽  
Marta L. DeDiego ◽  
Enrique Alvarez ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Subcellular Localization ◽  
Rna Binding ◽  
Cell Signalling ◽  
Bioinformatic Analysis ◽  
Binding Motif ◽  
Nuclear Localization Signals ◽  
N Protein ◽  
Localization Signal ◽  
Infected Cells

The coronavirus nucleocapsid (N) protein is a viral RNA-binding protein with multiple functions in terms of virus replication and modulating cell signalling pathways. N protein is composed of three distinct regions containing RNA-binding motif(s), and appropriate signals for modulating cell signalling. The subcellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) N protein was studied. In infected cells, SARS-CoV N protein localized exclusively to the cytoplasm. In contrast to the avian coronavirus N protein, overexpressed SARS-CoV N protein remained principally localized to the cytoplasm, with very few cells exhibiting nucleolar localization. Bioinformatic analysis and deletion mutagenesis coupled to confocal microscopy and live-cell imaging, revealed that SARS-CoV N protein regions I and III contained nuclear localization signals and region II contained a nucleolar retention signal. However, cytoplasmic localization was directed by region III and was the dominant localization signal in the protein.

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