Inline-tandem purification of viruses from cell lysate by agarose-based chromatography.

An efficient chromatography-based virus purification method has been developed and validated for the non-pathogenic infectious virus PRD1. Compared to the conventional method that consists of relatively time-consuming and labour-intensive precipitation and density gradient ultracentrifugation steps, the method developed here is performed in a single flow using tandem-coupled anion exchange and size exclusion chromatography (AIEX-SEC) columns. This inline approach helps to minimize the loss of virus in the process and streamlines time consumption, since no physical transfer of the sample is required between purification steps. In the development process, sample feed composition, dynamic binding capacity and elution conditions for the AIEX resin as well as different exclusion limits for SEC resins were optimized to achieve maximal yield of pure infectious viruses. Utilizing this new approach, a high-quality virus sample was produced from a lysate feed in 320 min with a total yield of 13 mg purified particles per litre of cell lysate, constituting a 3.5-fold yield increase as compared to the conventional method, without compromising the high specific infectivity of the product (6 × 1012 to 7 × 1012 pfu/mg of protein). The yield of infectious viruses of the lysate feed was 54%. The easy scalability of chromatography-based methods provide a direct route to industrial usage without any significant changes needed to be made to the purification regime. This is especially interesting as the method has high potential to be used for purification of various viruses and nanoparticles, including adenovirus.

Temperature dependency of virus and nanoparticle transport and retention in saturated porous media.

The influence of temperature on virus (PRD1 and ΦX174) and carboxyl-modified latex nanoparticle (50 and 100nm) attachment was examined in sand-packed columns under various physiochemical conditions. When the solution ionic strength (IS) equaled 10 and 30mM, the attachment rate coefficient (katt) increased up to 109% (p<0.0002) and the percentage of the sand surface area that contributed to attachment (Sf) increased up to 160% (p<0.002) when the temperature was increased from 4 to 20°C. Temperature effects at IS=10 and 30mM were also dependent on the system hydrodynamics; i.e., enhanced retention at a lower pore water velocity (0.1m/day). Conversely, this same temperature increase had a negligible influence on katt and Sf values when IS was 1mM or >50mM. An explanation for these observations was obtained from extended interaction energy calculations that considered nanoscale roughness and chemical heterogeneity on the sand surface. Interaction energy calculations demonstrated that the energy barrier to attachment in the primary minimum (∆Φa) decreased with increasing IS, chemical heterogeneity, and temperature, especially in the presence of small amounts of nanoscale roughness (e.g., roughness fraction of 0.05 and height of 20nm in the zone of influence). Temperature had a negligible effect on katt and Sf when the IS=1mM because of the large energy barrier, and at IS=50mM because of the absence of an energy barrier. Conversely, temperature had a large influence on katt and Sf when the IS was 10 and 30mM because of the presence of a small ∆Φa on sand with nanoscale roughness and a chemical (positive zeta potential) heterogeneity. This has large implications for setting parameters for the accurate modeling and transport prediction of virus and nanoparticle contaminants in ground water systems.

Effects of environmental and clinical interferents on the host capture efficiency of immobilized bacteriophages.

Bacteriophage-functionalized surfaces are a new class of advanced functional material and have been demonstrated to be applicable for use as antimicrobial surfaces in medical applications (e.g., indwelling medical devices or wound dressings) or as biosensors for bacterial capture and detection. However, the complex composition of many real life samples (e.g., blood, natural waters, etc.) can potentially interfere with the interaction of phage and its bacterial host, leading to a decline in the efficiency of the phage-functionalized surface. In this study, the bacterial capture efficiency of two model phage-functionalized surfaces was assessed in the presence of potential environmental and biomedical interferents. The two phage-bacteria systems used in this study are PRD1 with Salmonella Typhimurium and T4 with Escherichia coli. The potential interferents tested included humic and fulvic acids, natural groundwater, colloidal latex microspheres, host extracellular polymeric substances (EPS), albumin, fibrinogen, and human serum. EPS and human serum decreased the host capture efficiency for immobilized PRD1 and T4, and also impaired the infectivity of the nonimmobilized (planktonic) phage. Interestingly, humic and fulvic acids reduced the capture efficiency of T4-functionalized surfaces, even though they did not lead to inactivation of the suspended virions. Neither humic nor fulvic acids affected the capture efficiency of PRD1. These findings demonstrate the inadequacy of traditional phage selection methods (i.e., infectivity of suspended phage toward its host in clean buffer) for designing advanced functional materials and further highlight the importance of taking into account the environmental conditions in which the immobilized phage is expected to function.

Lipid-containing viruses: bacteriophage PRD1 assembly.

PRD1 is a tailless icosahedrally symmetric virus containing an internal lipid membrane beneath the protein capsid. Its linear dsDNA genome and covalently attached terminal proteins are delivered into the cell where replication occurs via a protein-primed mechanism. Extensive studies have been carried out to decipher the roles of the 37 viral proteins in PRD1 assembly, their association in virus particles and lately, especially the functioning of the unique packaging machinery that translocates the genome into the procapsid. These issues will be addressed in this chapter especially in the context of the structure of PRD1. We will also discuss the major challenges still to be addressed in PRD1 assembly.

Insights into the evolution of a complex virus from the crystal structure of vaccinia virus D13.

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.

Optimal preparation and purification of PRD1-like bacteriophages for use in environmental fate and transport studies.

Bacteriophages are bacterial viruses with unique characteristics that make them excellent surrogates for mammalian pathogenic viruses in environmental studies. Simple and reliable methodologies for isolation, detection, characterization and enumeration of somatic and F-specific bacteriophage are available in the literature. Limited information or methods are available for producing high-titer purified phage suspensions for studying microbial transport and survival in natural and engineered environments. This deficiency arises because most research on the production of high-titer phage suspensions was completed over half a century ago and more recent advances on these methods have not been compiled in a single publication. We present a review of the available methods and new data on the propagation, concentration and purification of two bacteriophage host systems (somatic PRD1/Salmonella thyphimurium and F-specific PR772/Escherichia coli) that are commonly utilized in laboratory and field-scale assessments of subsurface microbial transport and survival. The focus of the present study is to recommend the approach(es) that will ensure maximum bacteriophage yields while optimizing suspension purification (i.e. avoiding modification of surface charge of the phage capsids and/or inadvertent introduction of dissolved organic matter to the study system).

Tale of two spikes in bacteriophage PRD1.

Structural comparisons between bacteriophage PRD1 and adenovirus have revealed an evolutionary relationship that has contributed significantly to current ideas on virus phylogeny. However, the structural organization of the receptor-binding spike complex and how the different symmetry mismatches are mediated between the spike-complex proteins are not clear. We determined the architecture of the PRD1 spike complex by using electron microscopy and three-dimensional image reconstruction of a series of PRD1 mutants. We constructed an atomic model for the full-length P5 spike protein by using comparative modeling. P5 was shown to be bound directly to the penton base protein P31. P5 and the receptor-binding protein P2 form two separate spikes, interacting with each other near the capsid shell. P5, with a tumor necrosis factor-like head domain, may have been responsible for host recognition before capture of the current receptor-binding protein P2.

Structure of A197 from Sulfolobus turreted icosahedral virus: a crenarchaeal viral glycosyltransferase exhibiting the GT-A fold.

Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92 degrees C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the three domains of life. The 17,663-base-pair, circular, double-stranded DNA genome contains 36 potential open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. However, functional and evolutionary information may be suggested by a protein's three-dimensional structure. To this end, we have undertaken structural studies of the STIV proteome. Here we report our work on A197, the product of an STIV open reading frame. The structure of A197 reveals a GT-A fold that is common to many members of the glycosyltransferase superfamily. A197 possesses a canonical DXD motif and a putative catalytic base that are hallmarks of this family of enzymes, strongly suggesting a glycosyltransferase activity for A197. Potential roles for the putative glycosyltransferase activity of A197 and their evolutionary implications are discussed.

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.

The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture.

Comparisons of bacteriophage PRD1 and adenovirus protein structures and virion architectures have been instrumental in unraveling an evolutionary relationship and have led to a proposal of a phylogeny-based virus classification. The structure of the PRD1 spike protein P5 provides further insight into the evolution of viral proteins. The crystallized P5 fragment comprises two structural domains: a globular knob and a fibrous shaft. The head folds into a ten-stranded jelly roll beta barrel, which is structurally related to the tumor necrosis factor (TNF) and the PRD1 coat protein domains. The shaft domain is a structural counterpart to the adenovirus spike shaft. The structural relationships between PRD1, TNF, and adenovirus proteins suggest that the vertex proteins may have originated from an ancestral TNF-like jelly roll coat protein via a combination of gene duplication and deletion.

A quasi-atomic model of human adenovirus type 5 capsid.

Adenoviruses infect a wide range of vertebrates including humans. Their icosahedral capsids are composed of three major proteins: the trimeric hexon forms the facets and the penton, a noncovalent complex of the pentameric penton base and trimeric fibre proteins, is located at the 12 capsid vertices. Several proteins (IIIa, VI, VIII and IX) stabilise the capsid. We have obtained a 10 A resolution map of the human adenovirus 5 by image analysis from cryo-electron micrographs (cryoEMs). This map, in combination with the X-ray structures of the penton base and hexon, was used to build a quasi-atomic model of the arrangement of the two major capsid components and to analyse the hexon-hexon and hexon-penton interactions. The secondary proteins, notably VIII, were located by comparing cryoEM maps of native and pIX deletion mutant virions. Minor proteins IX and IIIa are located on the outside of the capsid, whereas protein VIII is organised with a T=2 lattice on the inner face of the capsid. The capsid organisation is compared with the known X-ray structure of bacteriophage PRD1.

Insights into assembly from structural analysis of bacteriophage PRD1.

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

Membrane structure and interactions with protein and DNA in bacteriophage PRD1.

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.

Phospholipid molecular species profiles of tectiviruses infecting Gram-negative and Gram-positive hosts.

The phospholipid (PL) molecular species compositions of bacteriophages PRD1 and Bam35 as well as their respective hosts were determined quantitatively using liquid chromatography/electrospray ionization mass-spectrometry (LC-ESI-MS) and backed up by gas-chromatographic/mass-spectrometric (GC-MS) analysis of the total fatty acids (FAs). The results showed that both viruses contain significantly more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membranes. Only modest differences in the molecular species composition of the viruses and their respective hosts were observed, indicating that the virus assembly process is relatively nonselective in respect of the fatty acid (FA) proportion of phospholipids (PL). These data indicate that the PL composition of these two viruses is largely, albeit not exclusively, determined by the availability of phospholipids in the host membrane.

Robust filtering and particle picking in micrograph images towards 3D reconstruction of purified proteins with cryo-electron microscopy.

In order to make a high resolution model of macromolecular structures from cryo-electron microscope (cryo-EM) raw images one has to be precise at every processing step from particle picking to 3D image reconstruction. In this paper we propose a collection of novel methods for filtering cryo-EM images and for automatic picking of particles. These methods have been developed for two cases: (1) when particles can be identified and (2) when particle are not distinguishable. The advantages of these methods are demonstrated in standard purified protein samples and to generalize them we do not use any ad hoc presumption of the geometry of the particle projections. We have also suggested a filtering method to increase the signal-to-noise (S/N) ratio which has proved to be useful for other levels of reconstruction, i.e., finding orientations and 3D model reconstruction.

Crystallization of the membrane-containing bacteriophage PRD1 in quartz capillaries by vapour diffusion.

Crystals of bacteriophage PRD1, a virus containing an internal lipid bilayer, have been grown in thin-walled quartz capillary tubes by vapour diffusion as a means of eliminating mechanical handling of the crystals during data collection. It has been found that the addition of polyethylene glycol 20 000 (PEG 20K) to the mother liquor that bathes the crystals allows far higher resolution diffraction intensities to be observed. Growing and treating the crystals in this way has produced a small number of crystals which are particularly amenable to X-ray diffraction analysis.

Diffraction quality crystals of PRD1, a 66-MDa dsDNA virus with an internal membrane.

It has proved difficult to obtain well diffracting single crystals of macromolecular complexes rich in lipid. We report here the path that has led to crystals of the bacteriophage PRD1, a particle containing approximately 2,000 protein subunits from 18 different protein species, around 10 of which are integral membrane proteins associated with a host-derived lipid bilayer of some 12,500 lipid molecules. These crystals are capable of diffracting X-rays to Bragg spacings below 4A. It is hoped that some lessons learned from PRD1 will be applicable to other lipidic systems and that these crystals will allow, as a proof of principle, the determination of the structure of the virus in terms of a detailed atomic model.

The X-ray crystal structure of P3, the major coat protein of the lipid-containing bacteriophage PRD1, at 1.65 A resolution.

P3 has been imaged with X-ray crystallography to reveal a trimeric molecule with strikingly similar characteristics to hexon, the major coat protein of adenovirus. The structure of native P3 has now been extended to 1.65 A resolution (R(work) = 19.0% and R(free) = 20.8%). The new high-resolution model shows that P3 forms crystals through hydrophobic patches solvated by 2-methyl-2,4-pentanediol molecules. It reveals details of how the molecule's high stability may be achieved through ordered solvent in addition to intra- and intersubunit interactions. Of particular importance is a 'puddle' at the top of the molecule containing a four-layer deep hydration shell that cross-links a complex structural feature formed by 'trimerization loops'. These loops also link subunits by extending over a neighbor to reach the third subunit in the trimer. As each subunit has two eight-stranded viral jelly rolls, the trimer has a pseudo-hexagonal shape to allow close packing in its 240 hexavalent capsid positions. Flexible regions in P3 facilitate these interactions within the capsid and with the underlying membrane. A selenometh-ionine P3 derivative, with which the structure was solved, has been refined to 2.2 A resolution (R(work) = 20.1% and R(free) = 22.8%). The derivatized molecule is essentially unchanged, although synchrotron radiation has the curious effect of causing it to rotate about its threefold axis. P3 is a second example of a trimeric 'double-barrel' protein that forms a stable building block with optimal shape for constructing a large icosahedral viral capsid. A major difference is that hexon has long variable loops that distinguish different adenovirus species. The short loops in P3 and the severe constraints of its various interactions explain why the PRD1 family has highly conserved coat proteins.