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1.
Int J Mol Sci ; 22(4)2021 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-33546457

RESUMO

Mononegavirales phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates N- and C-terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the Pneumoviridae P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. Pneumoviridae P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.


Assuntos
Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Pneumovirus/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Regulação Viral da Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Mononegavirais , Fosfoproteínas/genética , Pneumovirus/genética , Ligação Proteica , Dobramento de Proteína , Vírus Sincicial Respiratório Humano , Relação Estrutura-Atividade , Proteínas Virais/genética
2.
Viruses ; 12(11)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172032

RESUMO

The Asian tiger mosquito Aedes albopictus is a competent vector for several human arboviruses including dengue, chikungunya and Zika viruses. Mosquitoes also harbor insect-specific viruses (ISVs) that may modulate host physiology and potentially affect the transmission of viruses that are pathogenic to vertebrates, thus representing a potential tool for vector control strategies. In Ae. albopictus we identified a novel anphevirus (family Xinmoviridae; order Mononegavirales) provisionally designated here as Aedes albopictus anphevirus (AealbAV). AealbAV contains a ~12.4 kb genome that is highly divergent from currently known viruses but displays gene content and genomic organization typical of known anpheviruses. We identified AealbAV in several publicly available RNA-Seq datasets from different geographical regions both in laboratory colonies and field collected mosquitoes. Coding-complete genomes of AealbAV strains are highly similar worldwide (>96% nucleotide identity) and cluster according to the geographical origin of their hosts. AealbAV appears to be present in various body compartments and mosquito life stages, including eggs. We further detected AealbAV-derived vsiRNAs and vpiRNAs in publicly available miRNA-Seq libraries of Ae. albopictus and in samples experimentally coinfected with chikungunya virus. This suggests that AealbAV is targeted by the host RNA interference (RNAi) response, consistent with persistent virus replication. The discovery and characterization of AealbAV in Ae. albopictus will now allow us to identify its infection in mosquito populations and laboratory strains, and to assess its potential impact on Ae. albopictus physiology and ability to transmit arboviruses.


Assuntos
Aedes/virologia , Interações entre Hospedeiro e Microrganismos , Mononegavirais/classificação , Interferência de RNA , Animais , Vírus Chikungunya/genética , Coinfecção/virologia , Feminino , Genoma Viral , Vírus de Insetos/genética , Masculino , Mononegavirais/isolamento & purificação , Mononegavirais/fisiologia , Filogenia , Replicação Viral
3.
Arch Virol ; 165(12): 3023-3072, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32888050

RESUMO

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.


Assuntos
Mononegavirais/classificação , Terminologia como Assunto
4.
J Virol ; 94(22)2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-32847861

RESUMO

Mononegavirales, known as nonsegmented negative-sense (NNS) RNA viruses, are a class of pathogenic and sometimes deadly viruses that include rabies virus (RABV), human respiratory syncytial virus (HRSV), and Ebola virus (EBOV). Unfortunately, no effective vaccines and antiviral therapeutics against many Mononegavirales are currently available. Viral polymerases have been attractive and major antiviral therapeutic targets. Therefore, Mononegavirales polymerases have been extensively investigated for their structures and functions. Mononegavirales mimic RNA synthesis of their eukaryotic counterparts by utilizing multifunctional RNA polymerases to replicate entire viral genomes and transcribe viral mRNAs from individual viral genes as well as synthesize 5' methylated cap and 3' poly(A) tail of the transcribed viral mRNAs. The catalytic subunit large protein (L) and cofactor phosphoprotein (P) constitute the Mononegavirales polymerases. In this review, we discuss the shared and unique features of RNA synthesis, the monomeric multifunctional enzyme L, and the oligomeric multimodular adapter P of Mononegavirales We outline the structural analyses of the Mononegavirales polymerases since the first structure of the vesicular stomatitis virus (VSV) L protein determined in 2015 and highlight multiple high-resolution cryo-electron microscopy (cryo-EM) structures of the polymerases of Mononegavirales, namely, VSV, RABV, HRSV, human metapneumovirus (HMPV), and human parainfluenza virus (HPIV), that have been reported in recent months (2019 to 2020). We compare the structures of those polymerases grouped by virus family, illustrate the similarities and differences among those polymerases, and reveal the potential RNA synthesis mechanisms and models of highly conserved Mononegavirales We conclude by the discussion of remaining questions, evolutionary perspectives, and future directions.


Assuntos
Mononegavirais/enzimologia , Mononegavirais/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/química , Proteínas Virais/genética , Animais , Microscopia Crioeletrônica , Humanos , Metapneumovirus , Modelos Moleculares , Mononegavirais/classificação , Conformação Proteica , RNA Mensageiro , RNA Viral/genética , Vírus da Raiva , Vírus Sincicial Respiratório Humano , Vírus da Estomatite Vesicular Indiana/enzimologia , Vírus da Estomatite Vesicular Indiana/genética , Replicação Viral
6.
Arch Virol ; 164(4): 1233-1244, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30663023

RESUMO

In October 2018, the order Mononegavirales was amended by the establishment of three new families and three new genera, abolishment of two genera, and creation of 28 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Mononegavirais/classificação , Mononegavirais/genética , Mononegavirais/isolamento & purificação , Filogenia , Virologia/organização & administração
7.
Glycobiology ; 29(1): 2-21, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878112

RESUMO

Glycosylation is a biologically important protein modification process by which a carbohydrate chain is enzymatically added to a protein at a specific amino acid residue. This process plays roles in many cellular functions, including intracellular trafficking, cell-cell signaling, protein folding and receptor binding. While glycosylation is a common host cell process, it is utilized by many pathogens as well. Protein glycosylation is widely employed by viruses for both host invasion and evasion of host immune responses. Thus better understanding of viral glycosylation functions has potential applications for improved antiviral therapeutic and vaccine development. Here, we summarize our current knowledge on the broad biological functions of glycans for the Mononegavirales, an order of enveloped negative-sense single-stranded RNA viruses of high medical importance that includes Ebola, rabies, measles and Nipah viruses. We discuss glycobiological findings by genera in alphabetical order within each of eight Mononegavirales families, namely, the bornaviruses, filoviruses, mymonaviruses, nyamiviruses, paramyxoviruses, pneumoviruses, rhabdoviruses and sunviruses.


Assuntos
Glicoproteínas/metabolismo , Mononegavirais/metabolismo , Polissacarídeos/metabolismo , Proteínas Virais/metabolismo , Animais , Glicoproteínas/genética , Glicosilação , Humanos , Mononegavirais/genética , Polissacarídeos/genética , Proteínas Virais/genética
8.
Viruses ; 10(12)2018 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-30544818

RESUMO

The order Mononegavirales harbors numerous viruses of significant relevance to human health, including both established and emerging infections. Currently, vaccines are only available for a small subset of these viruses, and antiviral therapies remain limited. Being obligate cellular parasites, viruses must utilize the cellular machinery for their replication and spread. Therefore, targeting cellular pathways used by viruses can provide novel therapeutic approaches. One of the key challenges confronted by both hosts and viruses alike is the successful folding and maturation of proteins. In cells, this task is faced by cellular molecular chaperones, a group of conserved and abundant proteins that oversee protein folding and help maintain protein homeostasis. In this review, we summarize the current knowledge of how the Mononegavirales interact with cellular chaperones, highlight key gaps in our knowledge, and discuss the potential of chaperone inhibitors as antivirals.


Assuntos
Interações Hospedeiro-Patógeno , Chaperonas Moleculares/metabolismo , Mononegavirais/fisiologia , Antivirais/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Chaperonas Moleculares/antagonistas & inibidores , Mononegavirais/genética , Dobramento de Proteína , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/fisiologia , Replicação Viral/efeitos dos fármacos
9.
Nat Commun ; 9(1): 3057, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-30076298

RESUMO

Recent studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing. Here, using proteomics, confocal and single molecule super-resolution imaging, and infection under biosafety level-4 containment, we show that this nucleolar DDR pathway is targeted by infectious pathogens. We find that the matrix proteins of Hendra virus and Nipah virus, highly pathogenic viruses of the Henipavirus genus in the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1-Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhances henipavirus production. These data identify a mechanism for viral modulation of host cells by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intranucleolar function for proteins of any mononegavirus.


Assuntos
Nucléolo Celular/fisiologia , Nucléolo Celular/virologia , Dano ao DNA/fisiologia , Vírus Hendra/fisiologia , Vírus Nipah/fisiologia , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Células HeLa , Henipavirus/genética , Infecções por Henipavirus , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Mononegavirais/genética , Proteínas Nucleares/metabolismo , Nucleoproteínas/metabolismo , Proteômica , RNA Ribossômico/biossíntese , Proteínas Virais/metabolismo
10.
J Virol ; 92(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950416

RESUMO

Insect-specific viruses (ISVs) of the yellow fever mosquito Aedes aegypti have been demonstrated to modulate transmission of arboviruses such as dengue virus (DENV) and West Nile virus by the mosquito. The diversity and composition of the virome of A. aegypti, however, remains poorly understood. In this study, we characterized Aedes anphevirus (AeAV), a negative-sense RNA virus from the order Mononegavirales AeAV identified from Aedes cell lines was infectious to both A. aegypti and Aedes albopictus cells but not to three mammalian cell lines. To understand the incidence and genetic diversity of AeAV, we assembled 17 coding-complete and two partial genomes of AeAV from available transcriptome sequencing (RNA-Seq) data. AeAV appears to transmit vertically and be present in laboratory colonies, wild-caught mosquitoes, and cell lines worldwide. Phylogenetic analysis of AeAV strains indicates that as the A. aegypti mosquito has expanded into the Americas and Asia-Pacific, AeAV has evolved into monophyletic African, American, and Asia-Pacific lineages. The endosymbiotic bacterium Wolbachia pipientis restricts positive-sense RNA viruses in A. aegypti Reanalysis of a small RNA library of A. aegypti cells coinfected with AeAV and Wolbachia produces an abundant RNA interference (RNAi) response consistent with persistent virus replication. We found Wolbachia enhances replication of AeAV compared to a tetracycline-cleared cell line, and AeAV modestly reduces DENV replication in vitro The results from our study improve understanding of the diversity and evolution of the virome of A. aegypti and adds to previous evidence that shows Wolbachia does not restrict a range of negative-strand RNA viruses.IMPORTANCE The mosquito Aedes aegypti transmits a number of arthropod-borne viruses (arboviruses), such as dengue virus and Zika virus. Mosquitoes also harbor insect-specific viruses that may affect replication of pathogenic arboviruses in their body. Currently, however, there are only a few insect-specific viruses described from A. aegypti in the literature. Here, we characterize a novel negative-strand virus, AeAV. Meta-analysis of A. aegypti samples showed that it is present in A. aegypti mosquitoes worldwide and is vertically transmitted. Wolbachia-transinfected mosquitoes are currently being used in biocontrol, as they effectively block transmission of several positive-sense RNA viruses in mosquitoes. Our results demonstrate that Wolbachia enhances the replication of AeAV and modestly reduces dengue virus replication in a cell line model. This study expands our understanding of the virome in A. aegypti as well as providing insight into the complexity of the Wolbachia virus restriction phenotype.


Assuntos
Aedes/virologia , Perfilação da Expressão Gênica/métodos , Mononegavirais/fisiologia , Wolbachia/fisiologia , Aedes/microbiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Evolução Molecular , Genoma Viral , Especificidade de Hospedeiro , Humanos , Transmissão Vertical de Doenças Infecciosas/veterinária , Vírus de Insetos/classificação , Vírus de Insetos/fisiologia , Mononegavirais/classificação , Mosquitos Vetores/microbiologia , Mosquitos Vetores/virologia , Filogenia , Análise de Sequência de RNA , Células Vero , Replicação Viral
11.
Arch Virol ; 163(8): 2283-2294, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29637429

RESUMO

In 2018, the order Mononegavirales was expanded by inclusion of 1 new genus and 12 novel species. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.


Assuntos
Mononegavirais/classificação , Animais , Humanos , Mononegavirais/genética , Mononegavirais/isolamento & purificação , Infecções por Mononegavirales/veterinária , Infecções por Mononegavirales/virologia , Filogenia
12.
Viruses ; 9(5)2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28492506

RESUMO

The mononegaviral family Filoviridae has eight members assigned to three genera and seven species. Until now, genus and species demarcation were based on arbitrarily chosen filovirus genome sequence divergence values (≈50% for genera, ≈30% for species) and arbitrarily chosen phenotypic virus or virion characteristics. Here we report filovirus genome sequence-based taxon demarcation criteria using the publicly accessible PAirwise Sequencing Comparison (PASC) tool of the US National Center for Biotechnology Information (Bethesda, MD, USA). Comparison of all available filovirus genomes in GenBank using PASC revealed optimal genus demarcation at the 55-58% sequence diversity threshold range for genera and at the 23-36% sequence diversity threshold range for species. Because these thresholds do not change the current official filovirus classification, these values are now implemented as filovirus taxon demarcation criteria that may solely be used for filovirus classification in case additional data are absent. A near-complete, coding-complete, or complete filovirus genome sequence will now be required to allow official classification of any novel "filovirus." Classification of filoviruses into existing taxa or determining the need for novel taxa is now straightforward and could even become automated using a presented algorithm/flowchart rooted in RefSeq (type) sequences.


Assuntos
Filoviridae/classificação , Filoviridae/genética , Filogenia , Algoritmos , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Ebolavirus/classificação , Ebolavirus/genética , Variação Genética , Genoma Viral , Marburgvirus/classificação , Marburgvirus/genética , Mononegavirais/classificação , Mononegavirais/genética , Análise de Sequência de DNA , Design de Software , Especificidade da Espécie , Sequenciamento Completo do Genoma
13.
Arch Virol ; 162(8): 2493-2504, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28389807

RESUMO

In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Genoma Viral , Mononegavirais/classificação , Ordem dos Genes , Mononegavirais/genética , Filogenia , Especificidade da Espécie
14.
Uirusu ; 67(1): 69-78, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-29593155

RESUMO

Mononegaviruses are non-segmented negative-strand RNA viruses, and include measles, mumps, Marburg, and Ebola viruses. Measles virus and mumps virus, members of the family Paramyxoviridae, are immunotropic and neurotropic, respectively. Marburg virus and Ebola virus, members of the family Filoviridae, cause highly lethal hemorrhagic fever. In this paper, I summarize the recent structural and functional studies on the viral glycoproteins (GPs) of these viruses, which have shed light on virus entry and the humoral response. The structural and functional analyses of the interaction between viral GPs and receptors/antibodies also illuminate directions toward therapeutics against the viruses.


Assuntos
Anticorpos Antivirais/imunologia , Mononegavirais/patogenicidade , Internalização do Vírus , Glicoproteínas/química , Glicoproteínas/fisiologia , Humanos , Mononegavirais/imunologia , Proteínas Virais/química , Proteínas Virais/fisiologia
15.
J Invertebr Pathol ; 147: 37-50, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-27793741

RESUMO

Invertebrates are hosts to diverse RNA viruses that have all possible types of encapsidated genomes (positive, negative and ambisense single stranded RNA genomes, or a double stranded RNA genome). These viruses also differ markedly in virion morphology and genome structure. Invertebrate RNA viruses are present in three out of four currently recognized orders of RNA viruses: Mononegavirales, Nidovirales, and Picornavirales, and 10 out of 37 RNA virus families that have yet to be assigned to an order. This mini-review describes general properties of the taxonomic groups, which include invertebrate RNA viruses on the basis of their current classification by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Invertebrados/virologia , Mononegavirais/genética , Nidovirales/genética , Picornaviridae/genética , Animais , Interações Hospedeiro-Patógeno , Mononegavirais/classificação , Nidovirales/classificação , Filogenia , Picornaviridae/classificação
16.
Antiviral Res ; 134: 63-76, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27575793

RESUMO

Worldwide, respiratory syncytial virus (RSV) causes severe disease in infants, the elderly, and immunocompromised people. No vaccine or effective antiviral treatment is available. RSV is a member of the non-segmented, negative-strand (NNS) group of RNA viruses and relies on its RNA-dependent RNA polymerase to transcribe and replicate its genome. Because of its essential nature and unique properties, the RSV polymerase has proven to be a good target for antiviral drugs, with one compound, ALS-8176, having already achieved clinical proof-of-concept efficacy in a human challenge study. In this article, we first provide an overview of the role of the RSV polymerase in viral mRNA transcription and genome replication. We then review past and current approaches to inhibiting the RSV polymerase, including use of nucleoside analogs and non-nucleoside inhibitors. Finally, we consider polymerase inhibitors that hold promise for treating infections with other NNS RNA viruses, including measles and Ebola.


Assuntos
Antivirais/uso terapêutico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Mononegavirais/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Antivirais/farmacologia , Ensaios Clínicos como Assunto , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Humanos , Sarampo/tratamento farmacológico , Mononegavirais/enzimologia , Mononegavirais/genética , Nucleosídeos/agonistas , RNA Mensageiro , RNA Polimerase Dependente de RNA/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/enzimologia , Vírus Sincicial Respiratório Humano/genética , Transcrição Genética , Replicação Viral/efeitos dos fármacos
17.
J Mol Biol ; 428(17): 3467-82, 2016 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-27487481

RESUMO

The host innate immune system serves as the first line of defense against viral infections. Germline-encoded pattern recognition receptors detect molecular patterns associated with pathogens and activate innate immune responses. Of particular relevance to viral infections are those pattern recognition receptors that activate type I interferon responses, which establish an antiviral state. The order Mononegavirales is composed of viruses that possess single-stranded, non-segmented negative-sense (NNS) RNA genomes and are important human pathogens that consistently antagonize signaling related to type I interferon responses. NNS viruses have limited encoding capacity compared to many DNA viruses, and as a likely consequence, most open reading frames encode multifunctional viral proteins that interact with host factors in order to evade host cell defenses while promoting viral replication. In this review, we will discuss the molecular mechanisms of innate immune evasion by select NNS viruses. A greater understanding of these interactions will be critical in facilitating the development of effective therapeutics and viral countermeasures.


Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Tolerância Imunológica , Imunidade Inata , Mononegavirais/imunologia , Mononegavirais/patogenicidade , Animais , Humanos , Modelos Biológicos , Modelos Moleculares
18.
Arch Virol ; 161(8): 2351-60, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27216929

RESUMO

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).


Assuntos
Genoma Viral , Mononegavirais/classificação , Mononegavirais/genética , Filogenia
19.
Methods Mol Biol ; 1365: 357-72, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26498797

RESUMO

In this chapter, we describe laboratory protocols for rearing fish and a simple and efficient method of extracting and identifying pathogen and host proteins that may be involved in entry and replication of commercially important fish viruses. We have used the common carp (Cyprinus carpio L.) and goldfish (Cyprinus auratus) as a model system for studies of proteins involved in viral entry and replication. The chapter describes detailed protocols for maintenance of carp, cell culture, antibody purification of proteins, and use of electrospray-ionization mass spectrometry analysis to screen and identify cytoskeleton and other proteins that may be involved in viral infection and propagation in fish.


Assuntos
Carpas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Peixes/metabolismo , Carpa Dourada/metabolismo , Proteômica/métodos , Alphaherpesvirinae/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Carpas/virologia , Células Cultivadas , Proteínas do Citoesqueleto/imunologia , Proteínas de Peixes/imunologia , Carpa Dourada/virologia , Injeções , Mononegavirais/fisiologia , Internalização do Vírus , Replicação Viral
20.
Nat Commun ; 6: 8749, 2015 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-26549102

RESUMO

The L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site ((SAM)P) also contains a novel pocket ((NS)P) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the (SAM)P-adjoining site holding the nucleotides undergoing methylation ((SUB)P) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2'O and N7 positions, and also displays nucleotide triphosphatase activity.


Assuntos
Metapneumovirus/metabolismo , Capuzes de RNA/metabolismo , RNA/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Sítios de Ligação , Cromatografia em Camada Delgada , Cristalização , Cristalografia por Raios X , Metilação , Mononegavirais/metabolismo , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , Células Sf9 , Spodoptera , Proteínas Virais/química , Proteínas Virais/metabolismo
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