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1.
Front Microbiol ; 12: 737641, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34659168

RESUMO

Species of genus Shewanella are among the most frequently identified psychrotrophic bacteria. Here, we have studied the cellular properties, growth dynamics, and stress conditions of cold-active Shewanella strain #4, which was previously isolated from Baltic Sea ice. The cells are rod-shaped of ~2µm in length and 0.5µm in diameter, and they grow between 0 and 25°C, with an optimum at 15°C. The bacterium grows at a wide range of conditions, including 0.5-5.5% w/v NaCl (optimum 0.5-2% w/v NaCl), pH 5.5-10 (optimum pH 7.0), and up to 1mM hydrogen peroxide. In keeping with its adaptation to cold habitats, some polyunsaturated fatty acids, such as stearidonic acid (18:4n-3), eicosatetraenoic acid (20:4n-3), and eicosapentaenoic acid (20:5n-3), are produced at a higher level at low temperature. The genome is 4,456kb in size and has a GC content of 41.12%. Uniquely, strain #4 possesses genes for sialic acid metabolism and utilizes N-acetyl neuraminic acid as a carbon source. Interestingly, it also encodes for cytochrome c3 genes, which are known to facilitate environmental adaptation, including elevated temperatures and exposure to UV radiation. Phylogenetic analysis based on a consensus sequence of the seven 16S rRNA genes indicated that strain #4 belongs to genus Shewanella, closely associated with Shewanella aestuarii with a ~97% similarity, but with a low DNA-DNA hybridization (DDH) level of ~21%. However, average nucleotide identity (ANI) analysis defines strain #4 as a separate Shewanella species (ANI score=76). Further phylogenetic analysis based on the 92 most conserved genes places Shewanella strain #4 into a distinct phylogenetic clade with other cold-active marine Shewanella species. Considering the phylogenetic, phenotypic, and molecular characterization, we conclude that Shewanella strain #4 is a novel species and name it Shewanella glacialimarina sp. nov. TZS-4T, where glacialimarina means sea ice. Consequently, S. glacialimarina TZS-4T constitutes a promising model for studying transcriptional and translational regulation of cold-active metabolism.

2.
Nat Commun ; 10(1): 1184, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862777

RESUMO

The vertical double ß-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 Å resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single ß-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double ß-barrel MCPs.


Assuntos
Vírus de Archaea/fisiologia , Proteínas do Capsídeo/ultraestrutura , Vírus de DNA/fisiologia , Haloarcula/virologia , Montagem de Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Proteica em Folha beta , Multimerização Proteica , Vírion/ultraestrutura
3.
Environ Microbiol ; 21(6): 2129-2147, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30920125

RESUMO

The diversity of archaeal viruses is severely undersampled compared with that of viruses infecting bacteria and eukaryotes, limiting our understanding on their evolution and environmental impacts. Here, we describe the isolation and characterization of four new viruses infecting halophilic archaea from the saline Lake Retba, located close to Dakar on the coast of Senegal. Three of the viruses, HRPV10, HRPV11 and HRPV12, have enveloped pleomorphic virions and should belong to the family Pleolipoviridae, whereas the forth virus, HFTV1, has an icosahedral capsid and a long non-contractile tail, typical of bacterial and archaeal members of the order Caudovirales. Comparative genomic and phylogenomic analyses place HRPV10, HRPV11 and HRPV12 into the genus Betapleolipovirus, whereas HFTV1 appears to be most closely related to the unclassified Halorubrum virus HRTV-4. Differently from HRTV-4, HFTV1 encodes host-derived minichromosome maintenance helicase and PCNA homologues, which are likely to orchestrate its genome replication. HFTV1, the first archaeal virus isolated on a Haloferax strain, could also infect Halorubrum sp., albeit with an eightfold lower efficiency, whereas pleolipoviruses nearly exclusively infected autochthonous Halorubrum strains. Mapping of the metagenomic sequences from this environment to the genomes of isolated haloarchaeal viruses showed that these known viruses are underrepresented in the available viromes.


Assuntos
Vírus de Archaea/isolamento & purificação , Haloferax/virologia , Halorubrum/virologia , Lagos/virologia , Vírus de Archaea/classificação , Vírus de Archaea/genética , Metagenoma , Filogenia , Senegal , Vírion/classificação , Vírion/genética , Vírion/isolamento & purificação
4.
Nat Commun ; 10(1): 846, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783086

RESUMO

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.


Assuntos
Vírus de Archaea/química , Proteínas de Fusão de Membrana/química , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Halorubrum/virologia , Fusão de Membrana , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/química
5.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463970

RESUMO

Influenza A viruses (IAVs) are viral pathogens that cause epidemics and occasional pandemics of significant mortality. The generation of efficacious vaccines and antiviral drugs remains a challenge due to the rapid appearance of new influenza virus types and antigenic variants. Consequently, novel strategies for the prevention and treatment of IAV infections are needed, given the limitations of the presently available antivirals. Here, we used enzymatically produced IAV-specific double-stranded RNA (dsRNA) molecules and Giardia intestinalis Dicer for the generation of a swarm of small interfering RNA (siRNA) molecules. The siRNAs target multiple conserved genomic regions of the IAVs. In mammalian cells, the produced 25- to 27-nucleotide-long siRNA molecules are processed by endogenous Dicer into 21-nucleotide siRNAs and are thus designated Dicer-substrate siRNAs (DsiRNAs). We evaluated the efficacy of the above DsiRNA swarm at preventing IAV infections in human primary monocyte-derived macrophages and dendritic cells. The replication of different IAV strains, including avian influenza H5N1 and H7N9 viruses, was significantly inhibited by pretransfection of the cells with the IAV-specific DsiRNA swarm. Up to 7 orders of magnitude inhibition of viral RNA expression was observed, which led to a dramatic inhibition of IAV protein synthesis and virus production. The IAV-specific DsiRNA swarm inhibited virus replication directly through the RNA interference pathway although a weak induction of innate interferon responses was detected. Our results provide direct evidence for the feasibility of the siRNA strategy and the potency of DsiRNA swarms in the prevention and treatment of influenza, including the highly pathogenic avian influenza viruses.IMPORTANCE In spite of the enormous amount of research, influenza virus is still one of the major challenges for medical virology due to its capacity to generate new variants, which potentially lead to severe epidemics and pandemics. We demonstrated here that a swarm of small interfering RNA (siRNA) molecules, including more than 100 different antiviral RNA molecules targeting the most conserved regions of the influenza A virus genome, could efficiently inhibit the replication of all tested avian and seasonal influenza A variants in human primary monocyte-derived macrophages and dendritic cells. The wide antiviral spectrum makes the virus-specific siRNA swarm a potentially efficient treatment modality against both avian and seasonal influenza viruses.


Assuntos
Vírus da Influenza A/genética , Influenza Aviária/genética , RNA Interferente Pequeno/farmacologia , Animais , Antivirais/farmacologia , Aves , Linhagem Celular , RNA Helicases DEAD-box , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Cães , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Interferons/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Células Madin Darby de Rim Canino , Cultura Primária de Células , RNA Interferente Pequeno/genética , Ribonuclease III , Replicação Viral/efeitos dos fármacos
6.
Artigo em Inglês | MEDLINE | ID: mdl-30098552

RESUMO

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage ϕ6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of ϕ6. In addition, binding of ϕ6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of ~1 × 1013 PFU/mg of protein and ~65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with in-line light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.


Assuntos
Bacteriófago phi 6/isolamento & purificação , Fracionamento por Campo e Fluxo/métodos , Vírion/isolamento & purificação , Pseudomonas syringae/virologia , Ultracentrifugação , Ensaio de Placa Viral , Cultura de Vírus
7.
Res Microbiol ; 169(9): 500-504, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29772256

RESUMO

Certain pleomorphic archaeal viruses are highly infectious even at saturated salt. These viruses belong to the genus Betapleolipovirus of the recently described archaeal virus family Pleolipoviridae. Pleolipoviruses comprise single-stranded or double-stranded, circular or linear DNA genomes that share countless homologues among various archaeal genetic elements. Here we describe a new extremely halophilic betapleolipovirus, Halorubrum pleomorphic virus 9 (HRPV9), which has an integrase gene. We also identified new genes encoding minor pleolipoviral structural proteins. The studies on HRPV9 enhance our knowledge on pleolipoviruses, especially their reciprocal relatedness and relation to certain archaeal plasmids, proviruses and membrane vesicles.


Assuntos
Vírus de Archaea/enzimologia , Vírus de Archaea/genética , Integrases/genética , Salinidade , Vírus de Archaea/classificação , Vírus de Archaea/fisiologia , Vírus de DNA/genética , DNA Viral/genética , Genoma Viral , Fases de Leitura Aberta , Plasmídeos , Vírion
8.
Genome Announc ; 6(21)2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29798916

RESUMO

Phage Cr39582 was induced by mitomycin C from Pseudoalteromonas sp. strain Cr6751, isolated from a marine invertebrate gut. Pseudoalteromonas phage Cr39582 has 85% pairwise nucleotide identity with phage PM2 but lacks sequence homology in the spike protein. This report supports previous bioinformatic identification of corticoviral sequences within aquatic bacterial genomes.

9.
Sci Rep ; 8(1): 3771, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491457

RESUMO

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.


Assuntos
Bacteriófago phi X 174/química , Cristalografia por Raios X , Bacteriófago phi X 174/fisiologia , Microscopia Crioeletrônica , Escherichia coli/virologia
10.
Genes (Basel) ; 9(3)2018 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-29495629

RESUMO

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids.

11.
FEMS Microbiol Ecol ; 94(4)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29481638

RESUMO

Viruses are recognized as important actors in ocean ecology and biogeochemical cycles, but many details are not yet understood. We participated in a winter expedition to the Weddell Sea, Antarctica, to isolate viruses and to measure virus-like particle abundance (flow cytometry) in sea ice. We isolated 59 bacterial strains and the first four Antarctic sea-ice viruses known (PANV1, PANV2, OANV1 and OANV2), which grow in bacterial hosts belonging to the typical sea-ice genera Paraglaciecola and Octadecabacter. The viruses were specific for bacteria at the strain level, although OANV1 was able to infect strains from two different classes. Both PANV1 and PANV2 infected 11/15 isolated Paraglaciecola strains that had almost identical 16S rRNA gene sequences, but the plating efficiencies differed among the strains, whereas OANV1 infected 3/7 Octadecabacter and 1/15 Paraglaciecola strains and OANV2 1/7 Octadecabacter strains. All the phages were cold-active and able to infect their original host at 0°C and 4°C, but not at higher temperatures. The results showed that virus-host interactions can be very complex and that the viral community can also be dynamic in the winter-sea ice.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/crescimento & desenvolvimento , Camada de Gelo/microbiologia , Camada de Gelo/virologia , Proteobactérias/virologia , Regiões Antárticas , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Ecologia , Filogenia , Proteobactérias/classificação , Proteobactérias/genética , RNA Ribossômico 16S/genética , Estações do Ano , Água do Mar/microbiologia , Água do Mar/virologia
12.
Nat Rev Microbiol ; 15(12): 724-739, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29123227

RESUMO

One of the most prominent features of archaea is the extraordinary diversity of their DNA viruses. Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic viruses and represent unique virus families. The distinct nature of archaeal viruses also extends to the gene composition and architectures of their genomes and the properties of the proteins that they encode. Environmental research has revealed prominent roles of archaeal viruses in influencing microbial communities in ocean ecosystems, and recent metagenomic studies have uncovered new groups of archaeal viruses that infect extremophiles and mesophiles in diverse habitats. In this Review, we summarize recent advances in our understanding of the genomic and morphological diversity of archaeal viruses and the molecular biology of their life cycles and virus-host interactions, including interactions with archaeal CRISPR-Cas systems. We also examine the potential origins and evolution of archaeal viruses and discuss their place in the global virosphere.


Assuntos
Archaea/virologia , Vírus de Archaea/genética , Vírus de Archaea/fisiologia , Vírus de DNA/genética , Variação Genética , Genoma Viral
13.
J Gen Virol ; 98(12): 2916-2917, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29125455

RESUMO

Members of the family Pleolipoviridae (termed pleolipoviruses) are pseudo-spherical and pleomorphic archaeal viruses. The enveloped virion is a simple membrane vesicle, which encloses different types of DNA genomes of approximately 7-16 kbp (or kilonucleotides). Typically, virions contain a single type of transmembrane (spike) protein at the envelope and a single type of membrane protein, which is embedded in the envelope and located in the internal side of the membrane. All viruses infect extremely halophilic archaea in the class Halobacteria (phylum Euryarchaeota). Pleolipoviruses have a narrow host range and a persistent, non-lytic life cycle. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Pleolipoviridae which is available at www.ictv.global/report/pleolipoviridae.


Assuntos
Vírus/classificação , Genoma Viral , Especificidade de Hospedeiro , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fenômenos Fisiológicos Virais , Vírus/genética , Vírus/isolamento & purificação
14.
Extremophiles ; 21(6): 1119-1132, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29019077

RESUMO

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.


Assuntos
Vírus de Archaea/química , Cromatografia/métodos , Vírus de Archaea/metabolismo , Tolerância ao Sal
15.
Antivir Ther ; 22(7): 631-637, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28287396

RESUMO

BACKGROUND: Herpes simplex virus (HSV) is a common human pathogen. Despite current antivirals, it causes a significant medical burden. Drug resistant strains exist and they are especially prevalent in immunocompromised patients and in HSV eye infections. New treatment modalities are needed. METHODS: BALB/c mice were corneally infected with HSV and subsequently treated with a swarm of enzymatically created, Dicer-substrate small interfering RNA (siRNA) molecules that targeted the HSV gene UL29. Two infection models were used, one in which the infection was predominantly peripheral and another in which it spread to the central nervous system. Mouse survival, as well as viral spread, load, latency and peripheral shedding, was studied. RESULTS: The anti-HSV-UL29 siRNA swarm alleviated HSV infection symptoms, inhibited viral shedding and replication and had a favourable effect on mouse survival. CONCLUSIONS: Treatment with anti-HSV-UL29 siRNA swarm reduced symptoms and viral spread in HSV infection of mice and also inhibited local viral replication in mouse corneas.


Assuntos
Herpes Simples/virologia , RNA Interferente Pequeno/administração & dosagem , Simplexvirus/genética , Replicação Viral , Animais , Anti-Infecciosos Locais , Modelos Animais de Doenças , Feminino , Herpes Simples/mortalidade , Herpes Simples/terapia , Camundongos , RNA Interferente Pequeno/genética , Carga Viral , Ensaio de Placa Viral , Eliminação de Partículas Virais
16.
Viruses ; 9(2)2017 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-28218714

RESUMO

Members of the virus family Sphaerolipoviridae include both archaeal viruses and bacteriophages that possess a tailless icosahedral capsid with an internal membrane. The genera Alpha- and Betasphaerolipovirus comprise viruses that infect halophilic euryarchaea, whereas viruses of thermophilic Thermus bacteria belong to the genus Gammasphaerolipovirus. Both sequence-based and structural clustering of the major capsid proteins and ATPases of sphaerolipoviruses yield three distinct clades corresponding to these three genera. Conserved virion architectural principles observed in sphaerolipoviruses suggest that these viruses belong to the PRD1-adenovirus structural lineage. Here we focus on archaeal alphasphaerolipoviruses and their related putative proviruses. The highest sequence similarities among alphasphaerolipoviruses are observed in the core structural elements of their virions: the two major capsid proteins, the major membrane protein, and a putative packaging ATPase. A recently described tailless icosahedral haloarchaeal virus, Haloarcula californiae icosahedral virus 1 (HCIV-1), has a double-stranded DNA genome and an internal membrane lining the capsid. HCIV-1 shares significant similarities with the other tailless icosahedral internal membrane-containing haloarchaeal viruses of the family Sphaerolipoviridae. The proposal to include a new virus species, Haloarcula virus HCIV1, into the genus Alphasphaerolipovirus was submitted to the International Committee on Taxonomy of Viruses (ICTV) in 2016.


Assuntos
Vírus de Archaea/classificação , Vírus de Archaea/ultraestrutura , Bacteriófagos/classificação , Bacteriófagos/ultraestrutura , Filogenia , Vírion/ultraestrutura , Adenosina Trifosfatases/genética , Archaea/virologia , Vírus de Archaea/genética , Bacteriófagos/genética , Proteínas do Capsídeo/genética , Análise de Sequência de DNA , Thermus/virologia
17.
J Virol ; 91(8)2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28122979

RESUMO

Viral capsids ensure viral genome integrity by protecting the enclosed nucleic acids. Interactions between the genome and capsid and between individual capsid proteins (i.e., capsid architecture) are intimate and are expected to be characterized by strong evolutionary conservation. For this reason, a capsid structure-based viral classification has been proposed as a way to bring order to the viral universe. The seeming lack of sufficient sequence similarity to reproduce this classification has made it difficult to reject structural convergence as the basis for the classification. We reinvestigate whether the structure-based classification for viral coat proteins making icosahedral virus capsids is in fact supported by previously undetected sequence similarity. Since codon choices can influence nascent protein folding cotranslationally, we searched for both amino acid and nucleotide sequence similarity. To demonstrate the sensitivity of the approach, we identify a candidate gene for the pandoravirus capsid protein. We show that the structure-based classification is strongly supported by amino acid and also nucleotide sequence similarities, suggesting that the similarities are due to common descent. The correspondence between structure-based and sequence-based analyses of the same proteins shown here allow them to be used in future analyses of the relationship between linear sequence information and macromolecular function, as well as between linear sequence and protein folds.IMPORTANCE Viral capsids protect nucleic acid genomes, which in turn encode capsid proteins. This tight coupling of protein shell and nucleic acids, together with strong functional constraints on capsid protein folding and architecture, leads to the hypothesis that capsid protein-coding nucleotide sequences may retain signatures of ancient viral evolution. We have been able to show that this is indeed the case, using the major capsid proteins of viruses forming icosahedral capsids. Importantly, we detected similarity at the nucleotide level between capsid protein-coding regions from viruses infecting cells belonging to all three domains of life, reproducing a previously established structure-based classification of icosahedral viral capsids.


Assuntos
Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Vírus/classificação , Análise por Conglomerados , Conformação Proteica , Vírus/genética , Vírus/ultraestrutura
18.
Biochim Biophys Acta Gen Subj ; 1861(3): 664-672, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27993658

RESUMO

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host. These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle.


Assuntos
Membrana Celular/metabolismo , DNA Viral/genética , Adenosina Trifosfatases/metabolismo , Bacteriófago PRD1/genética , Capsídeo/metabolismo , DNA/genética , Genoma Viral/genética , Proteínas Virais/genética , Montagem de Vírus/genética
19.
FEBS J ; 284(2): 222-236, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27860276

RESUMO

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.


Assuntos
Antivirais/farmacologia , Fatores Imunológicos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Cinurenina/antagonistas & inibidores , Redes e Vias Metabólicas/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Feminino , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Indóis , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/metabolismo , Interferons/genética , Interferons/imunologia , Cinurenina/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/virologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Oxazóis/farmacologia , Oximas/farmacologia , Cultura Primária de Células , Pirróis/farmacologia , Sulfonamidas/farmacologia , Tiazóis/farmacologia , Transcriptoma , Triptofano/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
20.
RNA ; 23(1): 119-129, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803153

RESUMO

Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage φ6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the φ6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07-0.54 nm/sec), packaging could reach 4.62 nm/sec during the fast packaging phase.


Assuntos
Bacteriófago phi 6/fisiologia , RNA Viral/genética , Bacteriófago phi 6/genética , Técnicas In Vitro , Modelos Moleculares , Conformação de Ácido Nucleico , Dobramento de RNA , RNA Viral/química , Montagem de Vírus
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