scholarly journals Proteomics Analysis Unravels the Functional Repertoire of Coronavirus Nonstructural Protein 3

2008 ◽  
Vol 82 (11) ◽  
pp. 5279-5294 ◽  
Author(s):  
Benjamin W. Neuman ◽  
Jeremiah S. Joseph ◽  
Kumar S. Saikatendu ◽  
Pedro Serrano ◽  
Amarnath Chatterjee ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Rna Binding ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
Domain Architecture ◽  
Complete Characterization ◽  
Functional Domain ◽  
Metal Ion Binding ◽  
Nonstructural Protein 3

ABSTRACT Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

Ligand–Metal Ion Binding to Proteins: Investigation by ESI Mass Spectrometry

2005 ◽  
pp. 361-389 ◽  
Author(s):  
Noelle Potier ◽  
Hélène Rogniaux ◽  
Guillaume Chevreux ◽  
Alain Van Dorsselaer
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Esi Mass Spectrometry

scholarly journals Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1179 ◽  
Author(s):  
Anne Monette ◽  
Andrew J. Mouland
Keyword(s):  
Phase Separation ◽  
Metal Ion ◽  
Rna Binding ◽  
Copper Ions ◽  
Virus Assembly ◽  
Neurological Diseases ◽  
Meta Analysis ◽  
Metal Homeostasis ◽  
Virus Infections ◽  
As Stress

Liquid-liquid phase separation (LLPS) is a rapidly growing research focus due to numerous demonstrations that many cellular proteins phase-separate to form biomolecular condensates (BMCs) that nucleate membraneless organelles (MLOs). A growing repertoire of mechanisms supporting BMC formation, composition, dynamics, and functions are becoming elucidated. BMCs are now appreciated as required for several steps of gene regulation, while their deregulation promotes pathological aggregates, such as stress granules (SGs) and insoluble irreversible plaques that are hallmarks of neurodegenerative diseases. Treatment of BMC-related diseases will greatly benefit from identification of therapeutics preventing pathological aggregates while sparing BMCs required for cellular functions. Numerous viruses that block SG assembly also utilize or engineer BMCs for their replication. While BMC formation first depends on prion-like disordered protein domains (PrLDs), metal ion-controlled RNA-binding domains (RBDs) also orchestrate their formation. Virus replication and viral genomic RNA (vRNA) packaging dynamics involving nucleocapsid (NC) proteins and their orthologs rely on Zinc (Zn) availability, while virus morphology and infectivity are negatively influenced by excess Copper (Cu). While virus infections modify physiological metal homeostasis towards an increased copper to zinc ratio (Cu/Zn), how and why they do this remains elusive. Following our recent finding that pan-retroviruses employ Zn for NC-mediated LLPS for virus assembly, we present a pan-virus bioinformatics and literature meta-analysis study identifying metal-based mechanisms linking virus-induced BMCs to neurodegenerative disease processes. We discover that conserved degree and placement of PrLDs juxtaposing metal-regulated RBDs are associated with disease-causing prion-like proteins and are common features of viral proteins responsible for virus capsid assembly and structure. Virus infections both modulate gene expression of metalloproteins and interfere with metal homeostasis, representing an additional virus strategy impeding physiological and cellular antiviral responses. Our analyses reveal that metal-coordinated virus NC protein PrLDs initiate LLPS that nucleate pan-virus assembly and contribute to their persistence as cell-free infectious aerosol droplets. Virus aerosol droplets and insoluble neurological disease aggregates should be eliminated by physiological or environmental metals that outcompete PrLD-bound metals. While environmental metals can control virus spreading via aerosol droplets, therapeutic interference with metals or metalloproteins represent additional attractive avenues against pan-virus infection and virus-exacerbated neurological diseases.

scholarly journals Metal Ion Binding to the Amyloid β Monomer Studied by Native Top-Down FTICR Mass Spectrometry

2019 ◽  
Vol 30 (10) ◽  
pp. 2123-2134 ◽  
Author(s):  
Frederik Lermyte ◽  
James Everett ◽  
Yuko P. Y. Lam ◽  
Christopher A. Wootton ◽  
Jake Brooks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Amyloid Β ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Top Down ◽  
Fticr Mass Spectrometry

scholarly journals Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time-of-flight mass spectrometry

2009 ◽  
Vol 11 (7) ◽  
pp. 1738-1752 ◽  
Author(s):  
Ewald T.J. van den Bremer ◽  
Wim Jiskoot ◽  
Richard James ◽  
Geoffrey R. Moore ◽  
Colin Kleanthous ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Electrospray Ionization ◽  
Metal Ion ◽  
Time Of Flight ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Conformational Properties ◽  
Colicin E9

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication

Parasitology ◽  
2017 ◽  
Vol 145 (3) ◽  
pp. 313-325 ◽  
Author(s):  
AHMED THABET ◽  
JOHANNES SCHMIDT ◽  
SVEN BAUMANN ◽  
WALTHER HONSCHA ◽  
MARTIN VON BERGEN ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Toxoplasma Gondii ◽  
Metal Ion ◽  
Bioinformatic Analysis ◽  
Resistance Mechanisms ◽  
Host Cells ◽  
Intracellular Replication ◽  
Metal Ion Binding ◽  
Stable Isotope Labelling

SUMMARYMonensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.

scholarly journals Stress Granule Components G3BP1 and G3BP2 Play a Proviral Role Early in Chikungunya Virus Replication

2015 ◽  
Vol 89 (8) ◽  
pp. 4457-4469 ◽  
Author(s):  
Florine E. M. Scholte ◽  
Ali Tas ◽  
Irina C. Albulescu ◽  
Eva Žusinaite ◽  
Andres Merits ◽  
...  
Keyword(s):  
Viral Entry ◽  
Chikungunya Virus ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nonstructural Protein ◽  
Stress Granules ◽  
Genome Amplification ◽  
Translational Inhibition ◽  
Nonstructural Protein 3 ◽  
Transcription Complexes

ABSTRACTStress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored.IMPORTANCEChikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

scholarly journals A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Kelly E. Du Pont ◽  
Nicole R. Sexton ◽  
Martin McCullagh ◽  
Gregory D. Ebel ◽  
Brian J. Geiss
Keyword(s):  
Cell Culture ◽  
Viral Infection ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Energy Transduction ◽  
West Nile ◽  
Content Type ◽  
Nonstructural Protein 3

ABSTRACT The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo. We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus. IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo. This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

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