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1.
J Struct Biol ; 206(1): 119-127, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30825649

RESUMO

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus which can be involved in several central nervous system disorders such as encephalitis and meningitis. The VEEV genome codes for 4 non-structural proteins (nsP), of which nsP3 contains a Macro domain. Macro domains (MD) can be found as stand-alone proteins or embedded within larger proteins in viruses, bacteria and eukaryotes. Their most common feature is the binding of ADP-ribose (ADPr), while several macro domains act as ribosylation writers, erasers or readers. Alphavirus MD erase ribosylation but their precise contribution in viral replication is still under investigation. NMR-driven titration experiments of ADPr in solution with the VEEV macro domain (in apo- and complex state) show that it adopts a suitable conformation for ADPr binding. Specific experiments indicate that the flexibility of the loops ß5-α3 and α3-ß6 is critical for formation of the complex and assists a wrapping mechanism for ADPr binding. Furthermore, along with this sequence of events, the VEEV MD undergoes a conformational exchange process between the apo state and a low-populated "dark" conformational state.


Assuntos
Adenosina Difosfato Ribose/química , Vírus da Encefalite Equina Venezuelana/metabolismo , Simulação de Dinâmica Molecular , Domínios Proteicos , Proteínas não Estruturais Virais/química , Adenosina Difosfato Ribose/metabolismo , Animais , Vírus da Encefalite Equina Venezuelana/genética , Cavalos , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Ligação Proteica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
2.
PLoS Pathog ; 9(9): e1003631, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086133

RESUMO

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, N(TAIL), of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N(TAIL) domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N(TAIL) and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of N(TAIL) upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to N(TAIL) without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.


Assuntos
Vírus Hendra/química , Proteínas do Nucleocapsídeo/química , Fosfoproteínas/química , Cristalografia por Raios X , Vírus Hendra/genética , Vírus Hendra/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
3.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 6): 1589-603, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24914970

RESUMO

The structures of two constructs of the measles virus (MeV) phosphoprotein (P) multimerization domain (PMD) are reported and are compared with a third structure published recently by another group [Communie et al. (2013), J. Virol. 87, 7166-7169]. Although the three structures all have a tetrameric and parallel coiled-coil arrangement, structural comparison unveiled considerable differences in the quaternary structure and unveiled that the three structures suffer from significant structural deformation induced by intermolecular interactions within the crystal. These results show that crystal packing can bias conclusions about function and mechanism based on analysis of a single crystal structure, and they challenge to some extent the assumption according to which coiled-coil structures can be reliably predicted from the amino-acid sequence. Structural comparison also highlighted significant differences in the extent of disorder in the C-terminal region of each monomer. The differential flexibility of the C-terminal region is also supported by size-exclusion chromatography and small-angle X-ray scattering studies, which showed that MeV PMD exists in solution as a dynamic equilibrium between two tetramers of different compaction. Finally, the possible functional implications of the flexibility of the C-terminal region of PMD are discussed.


Assuntos
Biopolímeros/química , Vírus do Sarampo/química , Fosfoproteínas/química , Proteínas Virais/química , Dicroísmo Circular , Cristalografia por Raios X , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta
4.
Acta Crystallogr D Struct Biol ; 80(Pt 2): 113-122, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38265877

RESUMO

Phenuiviridae nucleoprotein is the main structural and functional component of the viral cycle, protecting the viral RNA and mediating the essential replication/transcription processes. The nucleoprotein (N) binds the RNA using its globular core and polymerizes through the N-terminus, which is presented as a highly flexible arm, as demonstrated in this article. The nucleoprotein exists in an `open' or a `closed' conformation. In the case of the closed conformation the flexible N-terminal arm folds over the RNA-binding cleft, preventing RNA adsorption. In the open conformation the arm is extended in such a way that both RNA adsorption and N polymerization are possible. In this article, single-crystal X-ray diffraction and small-angle X-ray scattering were used to study the N protein of Toscana virus complexed with a single-chain camelid antibody (VHH) and it is shown that in the presence of the antibody the nucleoprotein is unable to achieve a functional assembly to form a ribonucleoprotein complex.


Assuntos
Nucleoproteínas , Vírus da Febre do Flebótomo Napolitano , Nucleoproteínas/química , Vírus da Febre do Flebótomo Napolitano/genética , Vírus da Febre do Flebótomo Napolitano/metabolismo , Proteínas do Nucleocapsídeo/química , Modelos Moleculares , RNA Viral/química , RNA Viral/metabolismo
5.
PLoS Pathog ; 7(5): e1002059, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21637813

RESUMO

Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Šresolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.


Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Cristalização , Magnésio/metabolismo , Mutação/genética , Plasmídeos , Ligação Proteica , S-Adenosilmetionina/metabolismo
6.
Sci Adv ; 8(1): eabj7615, 2022 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-34985963

RESUMO

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)­competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo­electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

7.
Artigo em Inglês | MEDLINE | ID: mdl-21393853

RESUMO

To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Šresolution and crystal structure determination is in progress.


Assuntos
Metiltransferases/química , RNA Polimerase Dependente de RNA/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas não Estruturais Virais/química , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/isolamento & purificação , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação
8.
FEBS Open Bio ; 11(4): 1076-1083, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33534950

RESUMO

Arenaviruses are enveloped viruses containing a segmented, negative, and ambisense single-stranded RNA genome wrapped with a nucleoprotein (NP). The NP is the most abundant viral protein in infected cells and plays a critical role in both replication/transcription and virion assembly. The NP associates with RNA to form a ribonucleoprotein (RNP) complex, and this implies self-assembly while the exact structure of this polymer is not yet known. Here, we report a measurement of the full-length Mopeia virus NP by negative stain transmission electron microscopy. We observed RNP complex particles with diameter 15 ± 1 nm as well as symmetric circular heptamers of the same diameter, consistent with previous observations.


Assuntos
Arenavirus , Nucleoproteínas/química , Multimerização Proteica , Proteínas Virais/química , Sequência de Aminoácidos , Arenavirus/metabolismo , Modelos Moleculares , Nucleoproteínas/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas Recombinantes , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura
9.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 10): 1116-20, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20944244

RESUMO

The 2C protein, which is an essential ATPase and one of the most conserved proteins across the Picornaviridae family, is an emerging antiviral target for which structural and functional characterization remain elusive. Based on a distant relationship to helicases of small DNA viruses, piconavirus 2C proteins have been predicted to unwind double-stranded RNAs. Here, a terminally extended variant of the 2C protein from echovirus 30 has been studied by means of enzymatic activity assays, transmission electron microscopy, atomic force microscopy and dynamic light scattering. The transmission electron-microscopy technique showed the existence of ring-shaped particles with ∼12 nm external diameter. Image analysis revealed that these particles were hexameric and resembled those formed by superfamily 3 DNA virus helicases.


Assuntos
Vírus de DNA/fisiologia , Enterovirus Humano B/fisiologia , RNA Helicases/química , Proteínas Recombinantes/química , Proteínas Virais/química , Vírion/química , Técnicas In Vitro , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Conformação Proteica , Multimerização Proteica , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia Estrutural de Proteína , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Vírion/ultraestrutura
10.
J Virol ; 83(13): 6534-45, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19386706

RESUMO

Macro domains (also called "X domains") constitute a protein module family present in all kingdoms of life, including viruses of the Coronaviridae and Togaviridae families. Crystal structures of the macro domain from the Chikungunya virus (an "Old World" alphavirus) and the Venezuelan equine encephalitis virus (a "New World" alphavirus) were determined at resolutions of 1.65 and 2.30 A, respectively. These domains are active as adenosine di-phosphoribose 1''-phosphate phosphatases. Both the Chikungunya and the Venezuelan equine encephalitis virus macro domains are ADP-ribose binding modules, as revealed by structural and functional analysis. A single aspartic acid conserved through all macro domains is responsible for the specific binding of the adenine base. Sequence-unspecific binding to long, negatively charged polymers such as poly(ADP-ribose), DNA, and RNA is observed and attributed to positively charged patches outside of the active site pocket, as judged by mutagenesis and binding studies. The crystal structure of the Chikungunya virus macro domain with an RNA trimer shows a binding mode utilizing the same adenine-binding pocket as ADP-ribose, but avoiding the ADP-ribose 1''-phosphate phosphatase active site. This leaves the AMP binding site as the sole common feature in all macro domains.


Assuntos
Vírus Chikungunya/química , Vírus da Encefalite Equina Venezuelana/química , Proteínas não Estruturais Virais/química , Adenosina Difosfato Ribose/análogos & derivados , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Vírus Chikungunya/genética , Sequência Conservada , Vírus da Encefalite Equina Venezuelana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genética
11.
Viruses ; 12(7)2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32708976

RESUMO

Arenaviridae is a family of viruses harbouring important emerging pathogens belonging to the Bunyavirales order. Like in other segmented negative strand RNA viruses, the nucleoprotein (NP) is a major actor of the viral life cycle being both (i) the necessary co-factor of the polymerase present in the L protein, and (ii) the last line of defence of the viral genome (vRNA) by physically hiding its presence in the cytoplasm. The NP is also one of the major players interfering with the immune system. Several structural studies of NP have shown that it features two domains: a globular RNA binding domain (NP-core) in its N-terminal and an exonuclease domain (ExoN) in its C-terminal. Further studies have observed that significant conformational changes are necessary for RNA encapsidation. In this review we revisited the most recent structural and functional data available on Arenaviridae NP, compared to other Bunyavirales nucleoproteins and explored the structural and functional implications. We review the variety of structural motif extensions involved in NP-NP binding mode. We also evaluate the major functional implications of NP interactome and the role of ExoN, thus making the NP a target of choice for future vaccine and antiviral therapy.


Assuntos
Arenaviridae/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Montagem de Vírus , Arenaviridae/fisiologia , Proteínas do Nucleocapsídeo/fisiologia , Estrutura Terciária de Proteína
12.
Virology ; 529: 195-204, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30721816

RESUMO

The incidence of chikungunya virus (CHIKV) infection has increased dramatically in recent decades. Effective diagnostic methods must be available to optimize patient management. IgM-capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) is routinely used for the detection of specific CHIKV IgM. This method requires inactivated CHIKV viral lysate (VL). The use of viral bioparticles such as Virus-Like Particles (VLPs) and Pseudotyped-Particles (PPs) could represent an alternative to VL. Bioparticles performances were established by MAC-ELISA; physico-chemical characterizations were performed by field-flow fractionation (HF5) and confirmed by electron microscopy. Non-purified PPs give a detection signal higher than for VL. Results suggested that the signal difference observed in MAC-ELISA was probably due to the intrinsic antigenic properties of particles. The use of CHIKV bioparticles such as VLPs and PPs represents an attractive alternative to VL. Compared to VL and VLPs, non-purified PPs have proven to be more powerful antigens for specific IgM capture.


Assuntos
Anticorpos Antivirais/imunologia , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/fisiologia , Imunoglobulina M/imunologia , Testes Sorológicos/métodos , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Humanos , Sensibilidade e Especificidade
13.
Acta Crystallogr D Struct Biol ; 75(Pt 1): 8-15, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30644840

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a human pathogen responsible for a severe respiratory illness that emerged in 2012. Structural information about the proteins that constitute the viral particle is scarce. In order to contribute to a better understanding of the nucleoprotein (N) in charge of RNA genome encapsidation, the structure of the C-terminal domain of N from MERS-CoV obtained using single-crystal X-ray diffraction is reported here at 1.97 Šresolution. The molecule is present as a dimer in the crystal structure and this oligomerization state is confirmed in solution, as measured by additional methods including small-angle X-ray scattering measurements. Comparisons with the structures of the C-terminal domains of N from other coronaviruses reveals a high degree of structural conservation despite low sequence conservation, and differences in electrostatic potential at the surface of the protein.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/química , Nucleoproteínas/química , Multimerização Proteica , Humanos , Estrutura Molecular , Espalhamento a Baixo Ângulo , Eletricidade Estática , Proteínas Virais/química , Difração de Raios X
14.
J Mol Biol ; 431(12): 2283-2297, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30998933

RESUMO

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical "macro" (ßßαßßαßαßα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD-ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP-r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure-function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.


Assuntos
Nucleotídeos/metabolismo , RNA/metabolismo , Infecções por Togaviridae/virologia , Togaviridae/metabolismo , Proteínas não Estruturais Virais/metabolismo , Adenosina Difosfato Ribose/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Infecções por Togaviridae/metabolismo , Proteínas não Estruturais Virais/química
15.
Viruses ; 10(2)2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29385764

RESUMO

Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5'-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A-G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps.


Assuntos
Flavivirus/enzimologia , Flavivirus/genética , Nucleotídeos/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Domínio Catalítico , Modelos Moleculares , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Relação Estrutura-Atividade , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/genética , Replicação Viral
16.
Biomol NMR Assign ; 12(1): 31-35, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28875416

RESUMO

Macro domains are conserved protein domains found in eukaryotic organisms, bacteria, and archaea as well as in certain viruses. They consist of 130-190 amino acids and can bind ADP-ribose. Although the exact role of these domains is not fully understood, the conserved binding affinity for ADP-ribose indicates that this ligand is important for the function of the domain. Such a macro domain is also present in the non-structural protein 3 (nsP3) of Chikungunya Alphavirus (CHIKV) and consists of 160 amino acids. In this study we describe the high yield expression of the macro domain from CHIKV and its preliminary structural analysis via solution NMR spectroscopy. The macro domain seems to be folded in solution and an almost complete backbone assignment was achieved. In addition, the α/ß/α sandwich topology with 4 α-helices and 6 ß-strands was predicted by TALOS+.


Assuntos
Vírus Chikungunya , Ressonância Magnética Nuclear Biomolecular , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Domínios Proteicos
17.
Acta Crystallogr D Struct Biol ; 73(Pt 8): 641-649, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28777079

RESUMO

The Arenaviridae family is one of the two RNA viral families that encode a 3'-5' exonuclease in their genome. An exonuclease domain is found in the Arenaviridae nucleoprotein and targets dsRNA specifically. This domain is directly involved in suppression of innate immunity in the host cell. Like most phosphate-processing enzymes, it requires a divalent metal ion such as Mg2+ (or Mn2+) as a cofactor to catalyse nucleotide-cleavage and nucleotide-transfer reactions. On the other hand, calcium (Ca2+) inhibits this enzymatic activity, in spite of the fact that Mg2+ and Ca2+ present comparable binding affinities and biological availabilities. Here, the molecular and structural effects of the replacement of magnesium by calcium and its inhibition mechanism for phosphodiester cleavage, an essential reaction in the viral process of innate immunity suppression, are studied. Biochemical data and high-resolution structures of the Mopeia virus exonuclease domain complexed with each ion are reported for the first time. The consequences of the ion swap for the stability of the protein, the catalytic site and the functional role of a specific metal ion in enabling the catalytic cleavage of a dsRNA substrate are outlined.


Assuntos
Arenavirus/química , Arenavirus/enzimologia , Exonucleases/química , Proteínas do Nucleocapsídeo/química , Nucleoproteínas/química , Infecções por Arenaviridae/virologia , Arenavirus/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Domínio Catalítico , Cátions Bivalentes/metabolismo , Cristalização , Cristalografia por Raios X , Exonucleases/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/metabolismo , Nucleoproteínas/metabolismo , Domínios Proteicos , RNA Viral/metabolismo
18.
Acta Crystallogr D Struct Biol ; 73(Pt 8): 650-659, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28777080

RESUMO

Toscana virus (TOSV) is an arthropod-borne virus belonging to the Phlebovirus genus within the Bunyaviridae family. As in other bunyaviruses, the genome of TOSV is made up of three RNA segments. They are encapsidated by the nucleoprotein (N), which also plays an essential role in virus replication. To date, crystallographic structures of phlebovirus N have systematically revealed closed-ring organizations which do not fully match the filamentous organization of the ribonucleoprotein (RNP) complex observed by electron microscopy. In order to further bridge the gap between crystallographic data on N and observations of the RNP by electron microscopy, the structural organization of recombinant TOSV N was investigated by an integrative approach combining X-ray diffraction crystallography, transmission electron microscopy, small-angle X-ray scattering, size-exclusion chromatography and multi-angle laser light scattering. It was found that in solution TOSV N forms open oligomers consistent with the encapsidation mechanism of phlebovirus RNA.


Assuntos
Proteínas do Nucleocapsídeo/química , Nucleoproteínas/química , Vírus da Febre do Flebótomo Napolitano/química , Infecções por Bunyaviridae/virologia , Cristalografia por Raios X , Modelos Moleculares , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/ultraestrutura , Nucleoproteínas/metabolismo , Nucleoproteínas/ultraestrutura , Conformação Proteica , Multimerização Proteica , RNA Viral/metabolismo , Vírus da Febre do Flebótomo Napolitano/metabolismo , Espalhamento a Baixo Ângulo , Soluções , Difração de Raios X
20.
Acta Crystallogr D Struct Biol ; 72(Pt 2): 192-202, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26894667

RESUMO

The N protein of coronaviruses is a multifunctional protein that is organized into several domains. The N-terminal part is composed of an intrinsically disordered region (IDR) followed by a structured domain called the N-terminal domain (NTD). In this study, the structure determination of the N-terminal region of the MERS-CoV N protein via X-ray diffraction measurements is reported at a resolution of 2.4 Å. Since the first 30 amino acids were not resolved by X-ray diffraction, the structural study was completed by a SAXS experiment to propose a structural model including the IDR. This model presents the N-terminal region of the MERS-CoV as a monomer that displays structural features in common with other coronavirus NTDs.


Assuntos
Proteínas do Nucleocapsídeo/química , Cristalização , Cristalografia por Raios X , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Modelos Moleculares , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo
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