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1.
Acta Crystallogr D Struct Biol ; 77(Pt 7): 943-953, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34196620

RESUMO

Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Šresolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase and readily processes a variety of di-Ub linkages, in comparison with its Betacoronavirus counterparts, which have a narrower range of di-Ub activity but process both Ub and ISG15.


Assuntos
Infecções por Coronavirus/virologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Ubiquitina/metabolismo , Animais , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Suínos
2.
Sci Signal ; 14(689)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34131072

RESUMO

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.


Assuntos
COVID-19/virologia , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Manganês/metabolismo , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Capuzes de RNA/química , Capuzes de RNA/genética , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , Transdução de Sinais , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
3.
PLoS Pathog ; 15(1): e1007515, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629698

RESUMO

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.


Assuntos
Nairovirus/genética , Neoplasias Ovarianas/virologia , Peptídeo Hidrolases/genética , Cristalografia por Raios X/métodos , Enzimas Desubiquitinantes/metabolismo , Feminino , Variação Genética/genética , Genômica , Humanos , Nairovirus/patogenicidade , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Peptídeo Hidrolases/metabolismo , Filogenia , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional/genética , Proteólise , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
4.
ACS Infect Dis ; 4(9): 1316-1326, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-29856201

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is a widespread economically devastating disease caused by PRRS virus (PRRSV). First recognized in the late 1980s, PRRSV is known to undergo somatic mutations and high frequency viral recombination, which leads to many diverse viral strains. This includes differences within viral virulence factors, such as the viral ovarian tumor domain (vOTU) protease, also referred to as the papain-like protease 2. These proteases down-regulate innate immunity by deubiquitinating proteins targeted by the cell for further processing and potentially also acting against interferon-stimulated genes (ISGs). Recently, vOTUs from vaccine derivative Ingelvac PRRS modified live virus (MLV) and the highly pathogenic PRRSV strain JXwn06 were biochemically characterized, revealing a marked difference in activity toward K63 linked polyubiquitin chains and a limited preference for interferon-stimulated gene product 15 (ISG15) substrates. To extend our research, the vOTUs from NADC31 (low virulence) and SDSU73 (moderately virulent) were biochemically characterized using a myriad of ubiquitin and ISG15 related assays. The K63 polyubiquitin cleavage activity profiles of these vOTUs were found to track with the established pathogenesis of MLV, NADC31, SDSU73, and JXwn06 strains. Fascinatingly, NADC31 demonstrated significantly enhanced activity toward ISG15 substrates compared to its counterparts. Utilizing this information and strain-strain differences within the vOTU encoding region, sites were identified that can modulate K63 polyubiquitin and ISG15 cleavage activities. This information represents the basis for new tools to probe the role of vOTUs in the context of PRRSV pathogenesis.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Peptídeo Hidrolases/metabolismo , Poliubiquitina/metabolismo , Síndrome Respiratória e Reprodutiva Suína/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/enzimologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Fatores Reguladores de Interferon/química , Fatores Reguladores de Interferon/genética , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Poliubiquitina/química , Poliubiquitina/genética , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/química , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Alinhamento de Sequência , Suínos , Proteínas Virais/química , Proteínas Virais/genética , Virulência
5.
J Virol ; 91(23)2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28931677

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen that is the causative agent for Middle East respiratory syndrome (MERS). With MERS outbreaks resulting in over 35% fatalities and now spread to 27 countries, MERS-CoV poses a significant ongoing threat to global human health. As part of its viral genome, MERS-CoV encodes a papain-like protease (PLpro) that has been observed to act as a deubiquitinase and deISGylase to antagonize type I interferon (IFN-I) immune pathways. This activity is in addition to its viral polypeptide cleavage function. Although the overall impact of MERS-CoV PLpro function is observed to be essential, difficulty has been encountered in delineating the importance of its separate functions, particularly its deISGylase activity. As a result, the interface of MERS-CoV and human interferon-stimulated gene product 15 (hISG15) was probed with isothermal calorimetry, which suggests that the C-terminal domain of hISG15 is principally responsible for interactions. Subsequently, the structure of MERS-CoV PLpro was solved to 2.4 Å in complex with the C-terminal domain of hISG15. Utilizing this structural information, mutants were generated that lacked appreciable deISGylase activity but retained wild-type deubiquitinase and peptide cleavage activities. Hence, this provides a new platform for understanding viral deISGylase activity within MERS-CoV and other CoVs.IMPORTANCE Coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), encode a papain-like protease (PLpro) that possesses the ability to antagonize interferon immune pathways through the removal of ubiquitin and interferon-stimulated gene product 15 (ISG15) from target proteins. The lack of CoV proteases with attenuated deISGylase activity has been a key obstacle in delineating the impact between deubiquitinase and deISGylase activities on viral host evasion and pathogenesis. Here, biophysical techniques revealed that MERS-CoV PLpro chiefly engages human ISG15 through its C-terminal domain. The first structure of MERS-CoV PLpro in complex with this domain exposed the interface between these two entities. Employing these structural insights, mutations were employed to selectively remove deISGylase activity with no appreciable impact on its other deubiquitinase and peptide cleavage biochemical properties. Excitingly, this study introduces a new tool to probe the pathogenesis of MERS-CoV and related viruses through the removal of viral deISGylase activity.


Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Citocinas/química , Citocinas/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Ubiquitinas/química , Ubiquitinas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Calorimetria , Proteases 3C de Coronavírus , Cristalografia por Raios X , Enzimas Desubiquitinantes/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Interferons/antagonistas & inibidores , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mutação , Ubiquitina/metabolismo
6.
J Mol Biol ; 429(11): 1661-1683, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28438633

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode multifunctional papain-like proteases (PLPs) that have the ability to process the viral polyprotein to facilitate RNA replication and antagonize the host innate immune response. The latter function involves reversing the post-translational modification of cellular proteins conjugated with either ubiquitin (Ub) or Ub-like interferon-stimulated gene product 15 (ISG15). Ub is known to be highly conserved among eukaryotes, but surprisingly, ISG15 is highly divergent among animals. The ramifications of this sequence divergence to the recognition of ISG15 by coronavirus PLPs at a structural and biochemical level are poorly understood. Therefore, the activity of PLPs from SARS-CoV, MERS-CoV, and mouse hepatitis virus was evaluated against seven ISG15s originating from an assortment of animal species susceptible, and not, to certain coronavirus infections. Excitingly, our kinetic, thermodynamic, and structural analysis revealed an array of different preferences among PLPs. Included in these studies is the first insight into a coronavirus PLP's interface with ISG15 via SARS-CoV PLpro in complex with the principle binding domain of human ISG15 (hISG15) and mouse ISG15s (mISG15s). The first X-ray structure of the full-length mISG15 protein is also reported and highlights a unique, twisted hinge region of ISG15 that is not conserved in hISG15, suggesting a potential role in differential recognition. Taken together, this new information provides a structural and biochemical understanding of the distinct specificities among coronavirus PLPs observed and addresses a critical gap of how PLPs can interact with ISG15s from a wide variety of species.


Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Vírus da Hepatite Murina/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Ubiquitinas/química , Ubiquitinas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Cristalografia por Raios X , Humanos , Cinética , Camundongos , Ligação Proteica , Conformação Proteica
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