Purified Membrane-Containing Procapsids of Bacteriophage PRD1 Package the Viral Genome

Icosahedral-tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses translocate viral DNA into a preformed procapsid in an ATP-driven reaction by a packaging complex that operates at a portal vertex. A similar packaging system operates in the tailless dsDNA phage PRD1 (Tectiviridae family), except that there is an internal membrane vesicle in the procapsid. The unit-length linear dsDNA genome with covalently linked 5′-terminal proteins enters the procapsid through a unique vertex. Two small integral membrane proteins, P20 and P22, provide a conduit for DNA translocation. The packaging machinery also contains the packaging ATPase P9 and the packaging efficiency factor P6. Here we describe a method used to obtain purified packaging-competent PRD1 procapsids. The optimized in vitro packaging system allowed efficient packaging of defined DNA substrates. We determined that the genome terminal protein P8 is necessary for packaging and provided an estimation of the packaging rate.     

The unique vertex of bacterial virus PRD1 is connected to the viral internal membrane

Icosahedral double-stranded DNA (dsDNA) bacterial viruses are known to package their genomes into preformed procapsids via a unique portal vertex. Bacteriophage PRD1 differs from the more commonly known icosahedral dsDNA phages in that it contains an internal lipid membrane. The packaging of PRD1 is known to proceed via preformed empty capsids. Now, a unique vertex has been shown to exist in PRD1. We show in this study that this unique vertex extends to the virus internal membrane via two integral membrane proteins, P20 and P22. These small membrane proteins are necessary for the binding of the putative packaging ATPase P9, via another capsid protein, P6, to the virus particle.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

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.     

DNA packaging orders the membrane of bacteriophage PRD1.

Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat. Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3. The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice. The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane. The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density. The size of the coat does not change upon DNA packaging. Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase. Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses.     

Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery

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.     

Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica

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.     

A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

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.     

A snapshot of viral evolution from genome analysis of the tectiviridae family

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.     

Structure and assembly of bacteriophage PRD1, an Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5′ ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infectious virus.     

Structure and assembly of bacteriophage PRD1, and Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5' ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infections virus.     

Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications. The double-stranded DNA(dsDNA) viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules. The motor is geared by the hexameric RNA ring. An open washer displayed as hexametric string of phi29 motor ATPase has been reported. The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain. It was found that a free 5′-and 3′-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA. The above studies have inspired several applications in nanotechnology and nanomedicine. These applications include:(i) studies on the precision motor channels have led to their application in the single pore sensing;(ii) investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology; and(iii) the studies on the motor stoichiometry of homologous multi-subunits that subsequently have inspired the discovery of new methods in highly potent drug development. This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications. Given these advantages, more nanotechnological and biomedical applications using bacteriophage motor components are expected.     

Efficient DNA packaging of bacteriophage PRD1 requires the unique vertex protein P6

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.     

Real-time imaging of DNA ejection from single phage particles

Infection by tailed dsDNA phages is initiated by release of the viral DNA from the capsid and its polarized injection into the host. The driving force for the genome transport remains poorly defined. Among many hypothesis [1], it has been proposed that the internal pressure built up during packaging of the DNA in the capsid is responsible for its injection [2-4]. Whether the energy stored during packaging is sufficient to cause full DNA ejection or only to initiate the process was tested on phage T5 whose DNA (121,400 bp) can be released in vitro by mere interaction of the phage with its E. coli membrane receptor FhuA [5-7]. We present a fluorescence microscopy study investigating in real time the dynamics of DNA ejection from single T5 phages adsorbed onto a microfluidic cell. The ejected DNA was fluorescently stained, and its length was measured at different stages of the ejection after being stretched in a hydrodynamic flow. We conclude that DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75,000 bp/sec. The relevance of this stepwise ejection to the in vivo DNA transfer is discussed.     

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.     

Structural organization of DNA in chlorella viruses

Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm(-3). Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ～60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes.     

Minor proteins,mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.     

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.     

Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family TECTIVIRIDAE:     

The tailless icosahedral membrane virus PRD1 localizes the proteins involved in genome packaging and injection at a unique vertex

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.     

Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage phi29

During the assembly of many viruses, a powerful molecular motor compacts the genome into a preassembled capsid. Here, we present measurements of viral DNA packaging in bacteriophage phi29 using an improved optical tweezers method that allows DNA translocation to be measured from initiation to completion. This method allowed us to study the previously uncharacterized early stages of packaging and facilitated more accurate measurement of the length of DNA packaged. We measured the motor velocity versus load at near-zero filling and developed a ramped DNA stretching technique that allowed us to measure the velocity versus capsid filling at near-zero load. These measurements reveal that the motor can generate significantly higher velocities and forces than detected previously. Toward the end of packaging, the internal force resisting DNA confinement rises steeply, consistent with the trend predicted by many theoretical models. However, the force rises to a higher magnitude, particularly during the early stages of packaging, than predicted by models that assume coaxial inverse spooling of the DNA. This finding suggests that the DNA is not arranged in that conformation during the early stages of packaging and indicates that internal force is available to drive complete genome ejection in vitro. The maximum force exceeds 100 pN, which is about one-half that predicted to rupture the capsid shell.