Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus.

Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families.

Comparison of polymerase subunits from double-stranded RNA bacteriophages.

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage phi6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3' termini from the phi6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from phi6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the phi6-related bacteriophages phi8 and phi13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from phi6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3'-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit.

Combined EM/X-ray imaging yields a quasi-atomic model of the adenovirus-related bacteriophage PRD1 and shows key capsid and membrane interactions.

BACKGROUND: The dsDNA bacteriophage PRD1 has a membrane inside its icosahedral capsid. While its large size (66 MDa) hinders the study of the complete virion at atomic resolution, a 1.65-A crystallographic structure of its major coat protein, P3, is available. Cryo-electron microscopy (cryo-EM) and three-dimensional reconstruction have shown the capsid at 20-28 A resolution. Striking architectural similarities between PRD1 and the mammalian adenovirus indicate a common ancestor. RESULTS: The P3 atomic structure has been fitted into improved cryo-EM reconstructions for three types of PRD1 particles: the wild-type virion, a packaging mutant without DNA, and a P3-shell lacking the membrane and the vertices. Establishing the absolute EM scale was crucial for an accurate match. The resulting "quasi-atomic" models of the capsid define the residues involved in the major P3 interactions, within the quasi-equivalent interfaces and with the membrane, and show how these are altered upon DNA packaging. CONCLUSIONS: The new cryo-EM reconstructions reveal the structure of the PRD1 vertex and the concentric packing of DNA. The capsid is essentially unchanged upon DNA packaging, with alterations limited to those P3 residues involved in membrane contacts. These are restricted to a few of the N termini along the icosahedral edges in the empty particle; DNA packaging leads to a 4-fold increase in the number of contacts, including almost all copies of the N terminus and the loop between the two beta barrels. Analysis of the P3 residues in each quasi-equivalent interface suggests two sites for minor proteins in the capsid edges, analogous to those in adenovirus.

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Self-assembly of a viral molecular machine from purified protein and RNA constituents.

We present the assembly of the polymerase complex (procapsid) of a dsRNA virus from purified recombinant proteins. This molecular machine packages and replicates viral ssRNA genomic precursors in vitro. After addition of an external protein shell, these in vitro self-assembled viral core particles can penetrate the host plasma membrane and initiate a productive infection. Thus, a viral procapsid has been assembled and rendered infectious using purified components. Using this system, we have studied the mechanism of assembly of the common dsRNA virus shell and the incorporation of a symmetry mismatch within an icosahedral capsid. Our work demonstrates that this molecular machine, self-assembled under defined conditions in vitro, can function in its natural environment, the cell cytoplasm.

Crystallization and preliminary X-ray analysis of receptor-binding protein P2 of bacteriophage PRD1.

Bacteriophage PRD1 has remarkable structural similarities to adenovirus, but is unusual in containing a membrane beneath its icosahedral capsid. Its monomeric receptor-binding protein, P2, is part of a complex at each capsid vertex and so is the functional equivalent of adenovirus fiber. P2 has been crystallized by the "hanging-drop" method of vapor diffusion and two different crystal forms were obtained. Macroseeding, used to increase the size of the initial small needles, gave rod-shaped crystals. These grew to a size of 0.08 x 0.08 x 0.50 mm(3) and diffracted to 2.6 A resolution. They have the orthorhombic space group P222(1), with unit cell dimensions a = 137.8 A, b = 46.5 A, c = 136.4 A. A few single crystals of a second form were grown without seeding under slightly different conditions. A parallelepiped crystal (0.10 x 0.10 x 0.35 mm(3)), with space group C222(1) and unit cell dimensions a = 182.3 A, b = 204.8 A, c = 133.3 A, diffracted to 3.5 A resolution. A rotation function for the second form revealed that four monomers of P2 are related by a noncrystallographic twofold axis. The structure of P2 will reveal how this arrangement relates to the trimeric adenovirus fiber.

Assembly of bacteriophage PRD1 spike complex: role of the multidomain protein P5.

The spike structure of bacteriophage PRD1 is comprised of proteins P2, P5, and P31. It resembles the corresponding receptor-binding structure of adenoviruses. We show that purified recombinant protein P5 is an elongated (30 x 2.7 nm; R(h) = 5.5 nm), multidomain trimer which can slowly associate into nonamers. Cleavage of the 340 amino acid long P5 with collagenase yields 2 fragments. The larger, 205 amino acid long C-terminal fragment appears to contain the residues responsible for the trimerization of the protein, whereas the smaller N-terminal part mediates the interaction of P5 with the pentameric vertex protein P31 (24 x 2.5 nm, R(h) = 4.2 nm). In addition, the presence of the N-terminal sequence is required for the formation of the P5 nonamer. The results presented here suggest that P5 and P31 form an elongated adaptor complex at the 5-fold vertexes of the virion which anchors the adsorption protein P2 (21 x 2.5 nm; R(h) = 4.1 nm). Our results also suggest that the P5 trimer forms a substantial part of the viral spike shaft that was previously thought to be composed exclusively of protein P2.

A new mutant class, made by targeted mutagenesis, of phage PRD1 reveals that protein P5 connects the receptor binding protein to the vertex.

Phage PRD1 and adenovirus share a number of structural and functional similarities, one of which is the vertex organization at the fivefold-symmetry positions. We developed an in vitro mutagenesis system for the linear PRD1 genome in order to make targeted mutations. The role of protein P5 in the vertex structure was examined by this method. Mutation in gene V revealed that protein P5 is essential. The absence of P5 did not compromise the particle assembly or DNA packaging but led to a deficient vertex structure where the receptor binding protein P2, in addition to protein P5, was missing. P5(-) particles also lost their DNA upon purification. Based on this and previously published information we propose a spatial model for the spike structure at the vertices. This resembles to the corresponding structure in adenovirus.

Stable packaging of phage PRD1 DNA requires adsorption protein P2, which binds to the IncP plasmid-encoded conjugative transfer complex.

The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent K(d) of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor.

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.

Viral evolution revealed by bacteriophage PRD1 and human adenovirus coat protein structures.

The unusual bacteriophage PRD1 features a membrane beneath its icosahedral protein coat. The crystal structure of the major coat protein, P3, at 1.85 A resolution reveals a molecule with three interlocking subunits, each with two eight-stranded viral jelly rolls normal to the viral capsid, and putative membrane-interacting regions. Surprisingly, the P3 molecule closely resembles hexon, the equivalent protein in human adenovirus. Both viruses also have similar overall architecture, with identical capsid lattices and attachment proteins at their vertices. Although these two dsDNA viruses infect hosts from very different kingdoms, their striking similarities, from major coat protein through capsid architecture, strongly suggest their evolutionary relationship.

The complete genome sequence of PM2, the first lipid-containing bacterial virus To Be isolated.

Bacteriophage PM2 was isolated from the Pacific Ocean off the coast of Chile in the late 1960s. It was a new virus type, later classified as Corticoviridae, and also the first bacterial virus for which it was demonstrated that lipids are part of the virion structure. Here we report the determination and analysis of the 10, 079-bp circular dsDNA genome sequence. Noteworthy discoveries are the replication initiation system, which is related to the rolling circle mechanism described for phages such as φX174 and P2, and a 1.2-kb sequence that is similar to the maintenance region of a plasmid found in a marine Pseudoalteromonas sp. strain A28.

Purification and protein composition of PM2, the first lipid-containing bacterial virus to be isolated.

The marine, icosahedral bacteriophage PM2 was isolated in the late 1960s. It was the first phage for which lipids were firmly demonstrated to be part of the virion structure and it has been classified as the type organism of the Corticoviridae family. The host, Pseudoalteromonas espejiana BAL-31, belongs to a common group of marine bacteria. We developed a purification method producing virions with specific infectivity approximately as high as that of the lipid-containing phages PRD1 and φ6. The sensitivity of the virus to normally used purification media such as those containing sucrose is demonstrated. We also present an alternative host, a pseudoalteromonad, that allows enhanced purification of the virus under reduced salt conditions. We show, using N-terminal amino acid sequencing and comparison with the genomic sequence, that there are at least eight structural proteins in the infectious virus.

Bacteriophage PRD1 contains a labile receptor-binding structure at each vertex.

Bacteriophage PRD1 is a membrane-containing virus with an unexpected similarity to adenovirus. We mutagenized unassigned PRD1 genes to identify minor capsid proteins that could be structural or functional analogs to adenovirus proteins. We report here the identification of an amber mutant, sus525, in an essential PRD1 gene XXXI. The gene was cloned and the gene product was overexpressed and purified to near homogeneity. Analytical ultracentrifugation and gel filtration showed that P31 is a homopentamer of about 70 kDa. The protein was shown to be accessible on the virion surface and its absence in the sus525 particles led to the deficiency of two other viral coat proteins, protein P5 and the adsorption protein P2. Cryo-electron microscopy and image reconstruction of the sus525 particles indicate that these proteins are located on the capsid vertices, because in these particles the entire vertex structure was missing along with the peripentonal major capsid protein P3 trimers. Sus525 particles package DNA effectively but loose it upon purification. All of the PRD1 vertex structures are labile and potentially capable of mediating DNA delivery; this is in contrast to other dsDNA phages which employ a single vertex for packaging and delivery. We propose that this arises from a symmetry mismatch between protein P2 and the pentameric P31 in analogy to that between the adenovirus penton base and the receptor-binding spike.

A novel Raman spectrophotometric method for quantitative measurement of nucleoside triphosphate hydrolysis.

A novel spectrophotometric method, based upon Raman spectroscopy, has been developed for accurate quantitative determination of nucleoside triphosphate phosphohydrolase (NTPase) activity. The method relies upon simultaneous measurement in real time of the intensities of Raman marker bands diagnostic of the triphosphate (1115 cm(-1)) and diphosphate (1085 cm(-1)) moieties of the NTPase substrate and product, respectively. The reliability of the method is demonstrated for the NTPase-active RNA-packaging enzyme (protein P4) of bacteriophage phi6, for which comparative NTPase activities have been estimated independently by radiolabeling assays. The Raman-determined rate for adenosine triphosphate substrate (8.6 +/- 1.3 micromol x mg(-1) x min(-1) at 40 degrees C) is in good agreement with previous estimates. The versatility of the Raman method is demonstrated by its applicability to a variety of nucleotide substrates of P4, including the natural ribonucleoside triphosphates (ATP, GTP) and dideoxynucleoside triphosphates (ddATP, ddGTP). Advantages of the present protocol include conservative sample requirements (approximately 10(-6) g enzyme/protocol) and relative ease of data collection and analysis. The latter conveniences are particularly advantageous for the measurement of activation energies of phosphohydrolase activity.

Purification and characterization of the assembly factor P17 of the lipid-containing bacteriophage PRD1.

Assembly factors, proteins assisting the formation of viral structures, have been found in many viral systems. The gene encoding the assembly factor P17 of bacteriophage PRD1 has been cloned and expressed in Escherichia coli. P17 acts late in phage assembly, after capsid protein folding and multimerization, and sorting of membrane proteins has occurred. P17 has been purified to near homogeneity. It is a tetrameric protein displaying a rather high heat stability. The protein is largely in an alpha-helical conformation and possesses a putative leucine zipper which is not essential for protein function, as judged by in vitro mutagenesis and complementation analysis. Although heating does not cause structural changes in the conformation of the protein, the dissociation of the tetramer into smaller units is evident as diminished self-quenching of the fluorescently labeled P17. Similarly, dissociation of the tetramer is also obtained by dialysis of the protein against 6-M guanidine hydrochloride (GdnHCl) or 1% SDS. The reassembly of these smaller units upon cooling is evident from resonance energy transfer.

The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability.

IncP-type plasmids are broad-host-range conjugative plasmids. DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf). The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient. The Mpf complex acts as a receptor for IncP-specific phages such as PRD1. In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique. Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP. In addition it reduces the ability of the cells to accumulate K+. However, the Mpf complex does not dissipate the membrane voltage. The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.

Changes in host cell energetics in response to bacteriophage PRD1 DNA entry.

Double-stranded DNA bacteriophage PRD1 infects a variety of gram-negative bacteria harboring an IncP-type conjugative plasmid. The plasmid codes for the DNA transfer phage receptor complex in the cell envelope. Our goal was, by using a collection of mutant phage particles for which the variables are the DNA content and/or the presence of the receptor-binding protein, to obtain information on the energy requirements for DNA entry as well as on alterations in the cellular energetics taking place during the first stages of infection. We studied the fluxes of tetraphenylphosphonium (TPP+), phenyldicarbaundecaborane (PCB-), and K+ ions as well as ATP through the envelope of Salmonella typhimurium cells. The final level of the membrane voltage (delta psi) indicator TPP+ accumulated by the infected cells exceeds the initial level before the infection. Besides the effects on TPP+ accumulation, PRD1 induces the leakage of ATP and K+ from the cytosol. All these events were induced only by DNA-containing infectious particles and were cellular ATP and delta psi dependent. PRD1-caused changes in delta psi and in PCB- binding differ considerably from those observed in other bacteriophage infections studied. These results are in accordance with the presence of a specific channel engaged in phage PRD1 DNA transport.

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.

In vitro transcription of the double-stranded RNA bacteriophage phi 6 is influenced by purine NTPs and calcium.

The double-stranded RNA bacteriophage phi 6 contains a virion-associated RNA-dependent RNA polymerase complex. Removal of the virus envelope and the nucleocapsid surface protein, P8, reveals a nucleocapsid core particle (proteins P1, P2, P4, P7) which is the viral polymerase complex, capable of synthesizing RNA strands of positive polarity. The in vitro plus strand synthesis (transcription) reaction of the particle obtained from the mature virion was optimized and its activation and inactivation were investigated. Purine nucleoside triphosphates (NTPs), binding to a low-affinity binding site in the polymerase complex, activated plus strand synthesis. GTP was the preferred NTP, but dGTP, ddGTP, and the noncleavable analog GMP-PCP could also switch on transcription. This NTP-binding site is probably different from that of the unspecific viral NTPase found in protein P4 and also from that of the rNTP-specific RNA polymerase active site. Binding of purine NTPs was sufficient for the switch-on; hydrolysis of the NTP was not required. Besides nucleotides, divalent cations had an effect on phi 6 in vitro plus strand synthesis. Magnesium ions are required for the activity but calcium ions inhibit the reaction. Manganese ions are shown to dissipate the effect of magnesium and calcium ions, leading to uncontrolled, exceptionally high level plus strand synthesis.