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1.
Int J Mol Sci ; 21(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32707941

RESUMO

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.


Assuntos
Bacteriófagos/genética , Corticoviridae/genética , Cronobacter sakazakii/genética , DNA Viral/genética , Antígenos O/metabolismo , Fagos de Salmonella/genética , Salmonella/genética , Bacteriófagos/química , Bacteriófagos/metabolismo , Bacteriófagos/ultraestrutura , Classificação , Cronobacter sakazakii/virologia , Genoma Viral , Especificidade de Hospedeiro , Microscopia Eletrônica de Transmissão , Antígenos O/genética , Fases de Leitura Aberta , Proteômica , Salmonella/virologia , Análise de Sequência de DNA , Espectrometria de Massas em Tandem
2.
J Gen Virol ; 98(5): 888-889, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28581380

RESUMO

The Corticoviridae is a family of icosahedral, internal-membrane-containing viruses with double-stranded circular DNA genomes of approximately 10 kb. Only one species, Pseudoalteromonas virus PM2, has been recognized. Pseudoalteromonas virus PM2 infects Gram-negative bacteria and was isolated from seawater in 1968. Pseudoalteromonas virus PM2 is the first bacterial virus in which the presence of lipids in the virion has been demonstrated. Viral lipids are acquired selectively during virion assembly from the host cytoplasmic membrane. The outer protein capsid is an icosahedron with a pseudo T=21 symmetry. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Corticoviridae, which is available at www.ictv.global/report/corticoviridae.


Assuntos
Corticoviridae/química , Corticoviridae/classificação , Corticoviridae/genética , Corticoviridae/isolamento & purificação , Genoma Viral , Vírion/química , Vírion/classificação , Vírion/genética , Vírion/isolamento & purificação
3.
Radiats Biol Radioecol ; 54(4): 367-76, 2014.
Artigo em Russo | MEDLINE | ID: mdl-25775825

RESUMO

The objective of the study is elucidation of perspectives of 3,3'-diathylcarbocyaine application as a photosensitizer for curing viral infections by photodynamic therapy. Lipid-containing bacteriophage PM-2 of Pseudoalteromonas espejiana was used as a model. The testing was carried out at a special installation modeling photodynamic exposure conditions towards a non-fractionated phage lysate. 3,3'-DECC demonstrated a rapid photo-bleaching when added tothe phage lysate but not to water. The initial rate of PM-2 phage photoinactivation was proportional to the square concentration of the dye in the range of 0.5-9 µmol/L. This confirms a hypothesis that the dimer is the principal photochemically active form of the dye. An improved ability to form dimers was found in the dye in the phage lysate (10-folds better than in the water). The dye formed a stable adduct with the bacteriophage material. This adduct had an extinction maximum at λ(max) = 594 nm and demonstrated the properties of a polymer (sedimentation under a low-speed centrifugation).


Assuntos
Benzotiazóis/farmacologia , Carbocianinas/farmacologia , Corticoviridae/efeitos dos fármacos , Modelos Biológicos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Benzotiazóis/química , Benzotiazóis/uso terapêutico , Carbocianinas/química , Carbocianinas/uso terapêutico , Corticoviridae/efeitos da radiação , Dimerização , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Pseudoalteromonas/virologia , Viroses/tratamento farmacológico , Viroses/radioterapia
4.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 11): 2257-65, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24189238

RESUMO

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ~13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.


Assuntos
Substituição de Aminoácidos , Proteínas do Capsídeo/química , Corticoviridae/química , Modelos Moleculares , Espalhamento a Baixo Ângulo , Bromus/química , Bromus/ultraestrutura , Bromus/virologia , Proteínas do Capsídeo/ultraestrutura , Corticoviridae/ultraestrutura , Microscopia Crioeletrônica/métodos , Microscopia Crioeletrônica/tendências , Cristalização , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Vírus do Mosaico do Tabaco/química , Vírus do Mosaico do Tabaco/ultraestrutura , Vírus Satélite da Necrose do Tabaco/química , Vírus Satélite da Necrose do Tabaco/ultraestrutura , Tombusvirus/química , Tombusvirus/ultraestrutura
5.
Org Biomol Chem ; 11(32): 5300-9, 2013 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-23842892

RESUMO

It has been well documented that ß-carboline alkaloids, particularly the 9-methyl derivatives, are efficient photosensitizers. However, structure-activity relationships are missing and the photochemical mechanisms involved in the DNA photodamage still remain unknown. In the present work, we examined the capability of three 9-methyl-ß-carbolines (9-methyl-norharmane, 9-methyl-harmane and 9-methyl-harmine) to induce DNA damage upon UVA excitation at physiological pH. The type and extent of the damage was analyzed together with the photophysical and binding properties of the ß-carboline derivatives investigated. The results indicate that even at neutral pH most of the DNA damage is generated from the protonated form of the excited ß-carbolines in a type-I reaction. Oxidized purine residues are produced in high excess over oxidized pyrimidines, single-strand breaks and sites of base loss. In addition, the excited neutral form of the ß-carbolines is responsible for significant generation of cyclobutane pyrimidine dimers (CPDs) by triplet-triplet-energy transfer. In the case of 9-methyl-norharmane, the yield of CPDs is increased in D2O, probably due to less rapid protonation in the deuterated solvent.


Assuntos
Carbolinas/farmacologia , Corticoviridae/genética , Dano ao DNA/efeitos dos fármacos , DNA Viral/genética , DNA/genética , Fármacos Fotossensibilizantes/farmacologia , Animais , Carbolinas/química , Bovinos , Modelos Moleculares , Fármacos Fotossensibilizantes/química , Raios Ultravioleta
6.
Structure ; 19(7): 1011-20, 2011 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-21742267

RESUMO

The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double ß barrel "jelly-roll" subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Proteínas Recombinantes de Fusão/química , Vírus Vaccinia/química , Sequência de Aminoácidos , Bacteriófago PRD1/química , Evolução Biológica , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Corticoviridae/química , Cristalização , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Plasmídeos , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Vaccinia/virologia , Vírus Vaccinia/genética , Vírus Vaccinia/metabolismo
7.
Virology ; 405(1): 120-8, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20646729

RESUMO

Marine bacteriophage PM2 infects gram-negative Pseudoalteromonas species and is currently the only assigned member of the Corticoviridae family. The icosahedral protein shell covers an internal protein-rich phage membrane that encloses the highly supercoiled dsDNA genome. In this study we investigated PM2 entry into the host. Our results indicate that PM2 adsorption to the host is dependent on the intracellular ATP concentration, while genome penetration through the cytoplasmic membrane depends on the presence of millimolar concentrations of calcium ions in the medium. In the absence of Ca(2+) the infection is arrested at the entry stage but can be rescued by the addition of Ca(2+). Interestingly, PM2 entry induces abrupt cell lysis if the host outer membrane is not stabilized by divalent cations. Experimental data described in this study in combination with results obtained previously allowed us to propose a sequential model describing the entry of bacteriophage PM2 into the host cells.


Assuntos
Cálcio/metabolismo , Corticoviridae/fisiologia , Pseudoalteromonas/virologia , Internalização do Vírus , Trifosfato de Adenosina/metabolismo , Membrana Celular , Receptores de Superfície Celular , Fatores de Tempo
8.
Mol Cell ; 31(5): 749-61, 2008 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-18775333

RESUMO

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.


Assuntos
Evolução Biológica , Proteínas do Capsídeo/química , Corticoviridae/ultraestrutura , Lipídeos/química , Vírus/genética , Cálcio/metabolismo , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Corticoviridae/química , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Vírion/química , Vírion/ultraestrutura , Vírus/ultraestrutura
9.
J Bacteriol ; 190(4): 1298-307, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18083813

RESUMO

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.


Assuntos
Corticoviridae/genética , Pseudoalteromonas/virologia , Proteínas do Capsídeo/genética , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , DNA Circular/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/virologia , Vetores Genéticos/genética , Genoma Viral/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Fenótipo , Plasmídeos/genética , Pseudoalteromonas/genética , RNA de Transferência/genética , Água do Mar/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
J Struct Biol ; 161(2): 204-10, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18082422

RESUMO

There is a need for improved tools for labeling protein species within large macromolecular assemblies. Here we describe a method for the efficient selenomethionine labeling of the membrane-containing bacterial virus PM2 for structural studies. By examining potential host cells a strain was found which was auxotrophic for methionine, and by performing a multiparameter search of conditions it was possible to derive a robust protocol which simultaneously minimized the toxic effects of the selenomethionine, so that a reasonable virus yield was maintained, whilst still achieving essentially complete labeling. This has allowed us to fingerprint the protein constituents of the virus in a relatively low resolution electron density map. Such a technique can be adapted to other macromolecule complexes studied by X-ray crystallography.


Assuntos
Corticoviridae/química , Corticoviridae/ultraestrutura , Selenometionina/química , Cristalografia por Raios X , Métodos
11.
Mutat Res ; 625(1-2): 155-63, 2007 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17675188

RESUMO

Mitochondrial DNA (mtDNA) is assumed to be highly prone to damage by reactive oxygen species (ROS) because of its location in close proximity to the mitochondrial electron transport chain. Accordingly, mitochondrial oxidative DNA damage has been hypothesized to be responsible for various neurological diseases, ageing and cancer. Since 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most frequent oxidative base modifications, is removed from the mitochondrial genome by the glycosylase OGG1, the basal levels of this lesion are expected to be highly elevated in Ogg1(-/-) mice. To investigate this hypothesis, we have used a mtDNA relaxation assay in combination with various repair enzymes (Fpg, MutY, endonuclease III, endonuclease IV) to determine the average steady-state number of oxidative DNA modifications within intact (supercoiled) mtDNA from the livers of wild-type mice and those deficient in OGG1 and/or the Cockayne syndrome B (CSB) protein for mice aged up to 23 months. The levels of all types of oxidative modifications were found to be less than 12 per million base pairs, and the difference between wild-type and repair-deficient (Ogg1(-/-)/Csb(-/-)) mice was not significant. Thus, the increase of 8-oxoG caused by the repair deficiency in intact mtDNA is not much higher than in the nuclear DNA, i.e., not more than a few modifications per million base pairs. Based on these data, it is hypothesized that the load of oxidative base modifications in mtDNA is efficiently reduced during replication even in the absence of excision repair.


Assuntos
DNA Glicosilases/deficiência , Enzimas Reparadoras do DNA/deficiência , Reparo do DNA/fisiologia , DNA Mitocondrial/metabolismo , Guanosina/análogos & derivados , Animais , Corticoviridae/metabolismo , Dano ao DNA , DNA Glicosilases/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Mitocondrial/química , DNA Viral/química , DNA Viral/metabolismo , Feminino , Guanosina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Extratos Vegetais/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose
12.
BMC Genomics ; 8: 236, 2007 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-17634101

RESUMO

BACKGROUND: The origin and evolution of viruses is currently a heavily discussed issue. One element in this discussion is the innate viral "self" concept, which suggests that viral structures and functions can be divided into two categories. The first category consists of genetic determinants that are inherited from a viral ancestor and encode the viral "self". The second group consists of another set of structures and functions, the "nonself", which is interchangeable between different viruses and can be obtained via lateral gene transfer. Comparing the structures and sequences of the "self" elements, we have proposed that viruses can be grouped into lineages regardless of which domain of life (bacteria, archaea, eukarya) they infect. It has also been suggested that viruses are ancient and possibly predate modern cells. RESULTS: Here we identified thirteen putative prophages (viral genomes integrated into bacterial chromosome) closely related to the virulent icosahedral tailless lipid-containing bacteriophage PM2. Using the comparative genomics approach, we present evidence to support the viral "self" hypothesis and divide genes of the bacteriophage PM2 and related prophages into "self" and "nonself" categories. CONCLUSION: We show here that the previously proposed most conserved viral "self" determinants, the major coat protein and the packaging ATPase, were the only proteins that could be recognized in all detected corticoviral elements. We also argue here that the genes needed for viral genome replication, as well as for host cell lysis, belong to the "nonself" category of genes.Furthermore, we suggest that abundance of PM2-like viruses in the aquatic environment as well as their importance in the ecology of aquatic microorganisms might have been underestimated.


Assuntos
Corticoviridae/genética , Genoma Bacteriano/genética , Genômica/métodos , Prófagos/genética , Bactérias/genética , Bactérias/virologia , Proteínas do Capsídeo/genética , Bases de Dados de Proteínas , Evolução Molecular
13.
Mol Microbiol ; 64(6): 1635-48, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17555443

RESUMO

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.


Assuntos
Bacteriólise , Corticoviridae/fisiologia , Lipídeos/análise , Pseudoalteromonas/fisiologia , Pseudoalteromonas/virologia , Água do Mar/virologia , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Corticoviridae/química , Corticoviridae/genética , DNA/genética , Lisogenia , Dados de Sequência Molecular , Água do Mar/microbiologia , Proteínas Virais/química , Proteínas Virais/genética
14.
J Virol ; 80(18): 9270-8, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16940538

RESUMO

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.


Assuntos
Corticoviridae/genética , Elementos de DNA Transponíveis , Lipídeos/química , Mutagênese , Sequência de Aminoácidos , DNA/genética , Evolução Molecular , Genoma , Genoma Viral , Metabolismo dos Lipídeos , Dados de Sequência Molecular , Fases de Leitura Aberta , Fenótipo , Pseudoalteromonas
15.
Genetika ; 42(7): 898-903, 2006 Jul.
Artigo em Russo | MEDLINE | ID: mdl-16915919

RESUMO

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca2+ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 x 10(4)-2 x 10(5) infectious centers per microgram of PM2 DNA was developed.


Assuntos
Corticoviridae/genética , DNA Viral/genética , Pseudoalteromonas/genética , Transfecção/métodos
16.
Artigo em Inglês | MEDLINE | ID: mdl-16511151

RESUMO

PM2 (Corticoviridae) is a dsDNA bacteriophage which contains a lipid membrane beneath its icosahedral capsid. In this respect it resembles bacteriophage PRD1 (Tectiviridae), although it is not known whether the similarity extends to the detailed molecular architecture of the virus, for instance the fold of the major coat protein P2. Structural analysis of PM2 has been initiated and virus-derived P2 has been crystallized by sitting-nanodrop vapour diffusion. Crystals of P2 have been obtained in space group P2(1)2(1)2, with two trimers in the asymmetric unit and unit-cell parameters a = 171.1, b = 78.7, c = 130.1 A. The crystals diffract to 4 A resolution at the ESRF BM14 beamline (Grenoble, France) and the orientation of the non-crystallographic threefold axes, the spatial relationship between the two trimers and the packing of the trimers within the unit cell have been determined. The trimers form tightly packed layers consistent with the crystal morphology, possibly recapitulating aspects of the arrangement of subunits in the virus.


Assuntos
Proteínas do Capsídeo/química , Corticoviridae/química , Lipídeos/química , Cristalização , Cristalografia por Raios X
17.
J Microbiol ; 42(2): 99-102, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15357302

RESUMO

Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.


Assuntos
Corticoviridae/química , DNA Viral/análise , Endonucleases/metabolismo , Corticoviridae/genética , Corticoviridae/crescimento & desenvolvimento , Corticoviridae/isolamento & purificação , Criopreservação , Crioprotetores/farmacologia , DNA Circular/isolamento & purificação , DNA Circular/metabolismo , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Genoma Viral , Glicerol/farmacologia , Refrigeração , Trítio/metabolismo , Ensaio de Placa Viral
18.
Nat Struct Mol Biol ; 11(9): 850-6, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15286721

RESUMO

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.


Assuntos
Capsídeo/química , Membrana Celular/metabolismo , Corticoviridae/metabolismo , Vírion/química , Bacteriófagos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA Circular/química , Genoma , Genoma Viral , Processamento de Imagem Assistida por Computador , Metabolismo dos Lipídeos , Lipídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Pseudoalteromonas/virologia
19.
J Bacteriol ; 186(16): 5342-54, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15292135

RESUMO

The icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of the Corticoviridae family. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marine Pseudoalteromonas host has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.


Assuntos
Corticoviridae/fisiologia , Pseudoalteromonas/virologia , Bacteriófago PRD1/crescimento & desenvolvimento , Bacteriófago PRD1/fisiologia , Proteínas do Capsídeo/metabolismo , Membrana Celular/química , Corticoviridae/crescimento & desenvolvimento , DNA/metabolismo , DNA Viral/metabolismo , Gramicidina/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Peptidoglicano/metabolismo , Permeabilidade , Receptores Virais/fisiologia , Proteínas da Matriz Viral/metabolismo
20.
Antonie Van Leeuwenhoek ; 83(3): 223-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12776917

RESUMO

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report.


Assuntos
Corticoviridae/genética , Dano ao DNA , DNA Circular/isolamento & purificação , DNA Viral/isolamento & purificação , Endonucleases/metabolismo , Bioensaio , DNA Circular/metabolismo , DNA Circular/efeitos da radiação , DNA Viral/metabolismo , DNA Viral/efeitos da radiação , Polietilenoglicóis , Pseudoalteromonas/virologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Solventes , Raios Ultravioleta
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