RESUMEN

The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.

Asunto(s)

Bacteriófago PRD1/genética , Cápside , ADN Viral/genética , Genoma Viral , Microscopía de Fuerza Atómica , Nanoestructuras , Virión

RESUMEN

Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1-adenovirus lineage, characterized by a major capsid protein bearing two ß-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.

Asunto(s)

Proteínas de la Cápside/metabolismo , Virus ADN/genética , Flavobacterium/virología , Genoma Viral/genética , Bacteriófago PRD1/genética , Cápside , Virus ADN/clasificación , Virus ADN/aislamiento & purificación , ADN de Cadena Simple/genética , Lagos/virología , Conformación Proteica

RESUMEN

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.

Asunto(s)

Membrana Celular/metabolismo , ADN Viral/genética , Adenosina Trifosfatasas/metabolismo , Bacteriófago PRD1/genética , Cápside/metabolismo , ADN/genética , Genoma Viral/genética , Proteínas Virales/genética , Ensamble de Virus/genética

RESUMEN

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.

Asunto(s)

Bacteriófago PRD1/genética , Bacteriófago PRD1/ultraestructura , ADN Viral/genética , ADN/genética , Genoma Viral , Modelos Moleculares , Ensamble de Virus/genética , Cápside/química , Microscopía por Crioelectrón , ADN Viral/ultraestructura , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana , Membranas , Mutación/genética , Proteínas Virales , Virión/genética , Virión/ultraestructura

RESUMEN

Bacteria possess an intricate internal organization resembling that of the eukaryotes. The complexity is especially prominent at the bacterial cell poles, which are also known to be the preferable sites for some bacteriophages to infect. Bacteriophage PRD1 is a well-known model serving as an ideal system to study structures and functions of icosahedral internal membrane-containing viruses. Our aim was to analyze the localization and interactions of individual PRD1 proteins in its native host Escherichia coli. This was accomplished by constructing a vector library for production of fluorescent fusion proteins. Analysis of solubility and multimericity of the fusion proteins, as well as their localization in living cells by confocal microscopy, indicated that multimeric PRD1 proteins were prone to localize in the cell poles. Furthermore, PRD1 spike complex proteins P5 and P31, as fusion proteins, were shown to be functional in the virion assembly. In addition, they were shown to co-localize in the specific polar area of the cells, which might have a role in the multimerization and formation of viral protein complexes.

Asunto(s)

Bacteriófago PRD1/metabolismo , Escherichia coli/virología , Espacio Intracelular/virología , Proteínas Virales/metabolismo , Bacteriófago PRD1/genética , Transporte de Proteínas , Proteínas Virales/genética

Asunto(s)

Bacteriófago PRD1/genética , Genoma Viral/genética , Nanotubos/virología , Proteínas de la Cola de los Virus/metabolismo , Integración Viral/genética

RESUMEN

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

Asunto(s)

Bacteriófago PRD1/genética , Genoma Viral/genética , Nanotubos/virología , Proteínas de la Cola de los Virus/metabolismo , Integración Viral/genética , Bacteriófago PRD1/crecimiento & desarrollo , Bacteriófago PRD1/metabolismo , Cápside/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virología , ADN Viral/genética , Microscopía Electrónica , Salmonella typhimurium/virología , Integración Viral/fisiología

RESUMEN

Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5'-ends. In addition, they are DNA-binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp-1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA-binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp-1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.

Asunto(s)

Bacteriófagos/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Señales de Localización Nuclear , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Fagos de Bacillus/metabolismo , Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Bacteriófagos/genética , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Mutación Puntual , Proteínas Virales/genética

RESUMEN

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.

Asunto(s)

Fagos de Bacillus/fisiología , Bacteriófago PRD1/fisiología , Replicación del ADN/fisiología , ADN Viral/biosíntesis , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/virología , Bacteriófago PRD1/genética , Replicación del ADN/genética , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Genes Bacterianos , Genes Virales , Modelos Biológicos , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/genética , Replicación Viral/fisiología

RESUMEN

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Fagos de Bacillus/fisiología , Bacillus subtilis/metabolismo , Bacillus subtilis/virología , Proteínas Bacterianas/metabolismo , Replicación del ADN/fisiología , Replicación Viral/fisiología , Citoesqueleto de Actina/genética , Fagos de Bacillus/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Bacteriófago PRD1/genética , Bacteriófago PRD1/fisiología , Membrana Celular/metabolismo , Membrana Celular/virología , Replicación del ADN/genética , ADN Viral/biosíntesis , ADN Viral/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Mutación , Proteínas Virales/genética , Proteínas Virales/metabolismo , Acoplamiento Viral , Replicación Viral/genética

RESUMEN

Bacteriophage PRD1 is a tailless membrane-containing double-stranded (ds) DNA virus infecting a variety of Gram-negative bacteria. In order to affect cell lysis, like most dsDNA phages, PRD1 uses the holin-endolysin system. In this study, we identified two accessory lysis genes, XXXVI and XXXVII, coding for proteins P36 and P37, respectively. Using genetic complementation assays, we show that protein pair P36/P37 is a functional and interchangeable analogue of the Rz/Rz1 of bacteriophage lambda. Utilizing molecular biology, electrochemical as well as various microscopic techniques, we characterized the lysis phenotypes of PRD1 host cells infected with mutant viruses. Our results indicate that proteins P36 and P37 confer a competitive advantage to the phage by securing the efficient disruption of the infected cell and consequent release of the phage progeny under less favourable growth conditions. In concordance with prior data and the results obtained in this study, we propose a model explaining the role of Rz/Rz1-like proteins in the lysis process: Rz/Rz1 complexes transform the mechanical stress caused by the holin lesion at the CM to the OM leading to its disintegration. Finally, identification of the Rz/Rz1-like genes in PRD1 suggests that tailless icosahedral phages are involved in genetic trade with tailed bacteriophages.

Asunto(s)

Bacteriófago PRD1/enzimología , Endopeptidasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Bacteriólisis , Bacteriófago PRD1/genética , Bacteriófago lambda/enzimología , Bacteriófago lambda/genética , Coenzimas/farmacología , Endopeptidasas/genética , Escherichia coli K12/virología , Orden Génico , Prueba de Complementación Genética , Cloruro de Magnesio/farmacología , Modelos Biológicos , Datos de Secuencia Molecular , Salmonella typhimurium/citología , Salmonella typhimurium/virología , Alineación de Secuencia , Proteínas Virales/genética

RESUMEN

The viral lineage hypothesis predicting a common origin for viruses that infect hosts residing in different domains of life gains more support as data on viral structures accumulates. One such lineage is the PRD1-adenovirus lineage, which unites icosahedral dsDNA viruses with large facets and a double beta-barrel trimer coat protein. This lineage is represented by a number of viruses infecting bacteria and eukaryotes. However, only one member of the lineage, Sulfolobus turreted icosahedral virus, infecting a crenarchaeal host, has been identified in the domain Archaea. In this study we characterize the genomic sequences of two archaeal proviruses, TKV4 and MVV, integrated into the 5'- and 3'-distal regions of tRNA genes of the euryarchaeal species Thermococcus kodakaraensis KOD1 and Methanococcus voltae A3, respectively. Bioinformatic approaches allowed placement of TKV4 and MVV into the PRD1-adenovirus lineage, thus extending the lineage to the second archaeal phylum, Euryarchaeota.

Asunto(s)

Adenoviridae/clasificación , Adenoviridae/genética , Methanococcus/virología , Provirus/clasificación , Provirus/genética , Thermococcus/virología , Secuencia de Aminoácidos , Bacteriófago PRD1/genética , ADN de Archaea/genética , ADN Viral/genética , Modelos Moleculares , Datos de Secuencia Molecular , ARN de Transferencia/genética , Alineación de Secuencia , Sulfolobus

RESUMEN

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.

Asunto(s)

Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Empaquetamiento del ADN , Proteínas Virales/metabolismo , Ensamble de Virus , Bacteriófago PRD1/ultraestructura , Mutación , Salmonella enterica/ultraestructura , Salmonella enterica/virología , Proteínas Virales/genética , Virión/aislamiento & purificación , Virión/metabolismo , Virión/ultraestructura

RESUMEN

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.

Asunto(s)

Bacteriófago PRD1/química , Haloarcula/virología , Lípidos/análisis , Secuencia de Aminoácidos , Bacteriófago PRD1/genética , Proteínas de la Cápside/química , Genoma Viral , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas Estructurales Virales/análisis , Proteínas Estructurales Virales/genética

RESUMEN

PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only approximately 5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.

Asunto(s)

Bacteriólisis , Bacteriófago PRD1/fisiología , Endopeptidasas/metabolismo , Salmonella enterica/virología , Proteínas Virales/fisiología , Bacteriófago PRD1/genética , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Endopeptidasas/genética , Genes Virales , Prueba de Complementación Genética , Iones/metabolismo , Compuestos Onio , Compuestos Organofosforados , Potasio/metabolismo , Salmonella enterica/genética , Salmonella enterica/fisiología , Proteínas Virales/genética

RESUMEN

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.

Asunto(s)

Genoma Viral , Tectiviridae/genética , Secuencia de Aminoácidos , Bacteriófago PRD1/genética , Bacteriófagos/metabolismo , Secuencia de Bases , Membrana Celular/metabolismo , Biología Computacional , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Bases de Datos de Proteínas , Escherichia coli/metabolismo , Evolución Molecular , Prueba de Complementación Genética , Lípidos/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Operón , Filogenia , Plásmidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Ácido Nucleico , Programas Informáticos

RESUMEN

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.

Asunto(s)

Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Empaquetamiento del ADN , ADN Viral/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cápside/metabolismo , ADN Viral/química , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis , Alineación de Secuencia , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Membranes are essential for selectively controlling the passage of molecules in and out of cells and mediating the response of cells to their environment. Biological membranes and their associated proteins present considerable difficulties for structural analysis. Although enveloped viruses have been imaged at about 9 A resolution by cryo-electron microscopy and image reconstruction, no detailed crystallographic structure of a membrane system has been described. The structure of the bacteriophage PRD1 particle, determined by X-ray crystallography at about 4 A resolution, allows the first detailed analysis of a membrane-containing virus. The architecture of the viral capsid and its implications for virus assembly are presented in the accompanying paper. Here we show that the electron density also reveals the icosahedral lipid bilayer, beneath the protein capsid, enveloping the viral DNA. The viral membrane contains about 26,000 lipid molecules asymmetrically distributed between the membrane leaflets. The inner leaflet is composed predominantly of zwitterionic phosphatidylethanolamine molecules, facilitating a very close interaction with the viral DNA, which we estimate to be packaged to a pressure of about 45 atm, factors that are likely to be important during membrane-mediated DNA translocation into the host cell. In contrast, the outer leaflet is enriched in phosphatidylglycerol and cardiolipin, which show a marked lateral segregation within the icosahedral asymmetric unit. In addition, the lipid headgroups show a surprising degree of order.

Asunto(s)

Bacteriófago PRD1/química , Bacteriófago PRD1/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , ADN Viral/metabolismo , Proteínas Virales/metabolismo , Bacteriófago PRD1/genética , Cápside/química , Cápside/metabolismo , Cristalografía por Rayos X , ADN Viral/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas Virales/química , Ensamble de Virus

RESUMEN

The icosahedral membrane-containing double-stranded DNA bacteriophage PRD1 has a labile receptor binding spike complex at the vertices. This complex, which is analogous to that of adenovirus, is formed of the penton protein P31, the spike protein P5, and the receptor binding protein P2. Upon infection, the internal phage membrane transforms into a tubular structure that protrudes through a vertex and penetrates the cell envelope for DNA injection. We describe here a new class of PRD1 mutants lacking virion-associated integral membrane protein P16. P16 links the spike complex to the viral membrane and is necessary for spike stability. We also show that the unique vertex used for DNA packaging is intact in the P16-deficient particle, indicating that the 11 adsorption vertices and the 1 portal vertex are functionally and structurally distinct.

Asunto(s)

Bacteriófago PRD1/fisiología , Proteínas de la Matriz Viral/fisiología , Secuencia de Aminoácidos , Bacteriófago PRD1/genética , Bacteriófago PRD1/patogenicidad , Bacteriófago PRD1/ultraestructura , Genes Virales , Prueba de Complementación Genética , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Fenotipo , Receptores Virales/fisiología , Salmonella enterica/virología , Proteínas de la Matriz Viral/genética

RESUMEN

Extra- and intracellular viruses in the biosphere outnumber their cellular hosts by at least one order of magnitude. How is this enormous domain of viruses organized? Sampling of the virosphere has been scarce and focused on viruses infecting humans, cultivated plants, and animals as well as those infecting well-studied bacteria. It has been relatively easy to cluster closely related viruses based on their genome sequences. However, it has been impossible to establish long-range evolutionary relationships as sequence homology diminishes. Recent advances in the evaluation of virus architecture by high-resolution structural analysis and elucidation of viral functions have allowed new opportunities for establishment of possible long-range phylogenic relationships-virus lineages. Here, we use a genomic approach to investigate a proposed virus lineage formed by bacteriophage PRD1, infecting gram-negative bacteria, and human adenovirus. The new member of this proposed lineage, bacteriophage Bam35, is morphologically indistinguishable from PRD1. It infects gram-positive hosts that evolutionarily separated from gram-negative bacteria more than one billion years ago. For example, it can be inferred from structural analysis of the coat protein sequence that the fold is very similar to that of PRD1. This and other observations made here support the idea that a common early ancestor for Bam35, PRD1, and adenoviruses existed.

Asunto(s)

Fagos de Bacillus/genética , Bacillus thuringiensis/virología , Bacteriófago PRD1/genética , Genoma Viral , Fagos de Bacillus/aislamiento & purificación , Fagos de Bacillus/patogenicidad , Bacteriófago PRD1/aislamiento & purificación , Bacteriófago PRD1/patogenicidad , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Evolución Molecular , Microscopía Electrónica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia , Proteínas Virales/análisis