Bacteriophage PP7 Research Articles

Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

BackgroundTo be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves.ResultsHere we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs) displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C.ConclusionsVLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration

Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34–60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5–5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.

Immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus L2 epitope, on virus-like particles of the RNA bacteriophage PP7

The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high-titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format.

Examination of the Adsorption of Large Biological Molecules to Anion Exchange Surfaces Using Surface Plasmon Resonance

Separation of viruses and other contaminants from protein therapeutics using anion exchange membrane adsorbers is a successful new approach to viral clearance; however, the fundamental phenomena that control performance are not well understood. For example, the kinetics of adsorption to the anion exchange surface may limit clearance, but has yet to be characterized experimentally and mathematically. In the present study, surface plasmon resonance was used to determine the adsorption kinetics for five large biological molecules: phage PP7, phage ΦX174, phage PR772, thyroglobulin, and DNA. Rate constants were incorporated into a kinetic model of chromatography to illustrate the impact on performance.

Particle concentration measurement of virus samples using electrospray differential mobility analysis and quantitative amino acid analysis

Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and ϕX174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed.

Combined Use of MS2 and PP7 Coat Fusions Shows that TIA‐1 Dominates hnRNP A1 for K‐SAM Exon Splicing Control

Splicing of the FGFR2 K-SAM exon is repressed by hnRNP A1 bound to the exon and activated by TIA-1 bound to the downstream intron. Both proteins are expressed similarly by cells whether they splice the exon or not, so it is important to know which one is dominant. To answer this question, we used bacteriophage PP7 and bacteriophage MS2 coat fusions to tether hnRNP A1 and TIA-1 to distinct sites on the same pre-mRNA molecule. hnRNP A1 fused to one coat protein was tethered to a K-SAM exon containing the corresponding coat protein's binding site. TIA-1 fused to the other coat protein was tethered to the downstream intron containing that coat protein's binding site. This led to efficient K-SAM exon splicing. Our results show that TIA-1 is dominant for K-SAM exon splicing control and validate the combined use of PP7 and MS2 coat proteins for studying posttranscriptional events.

Another handle for RNA

The structure of the PP7 bacteriophage coat protein revealed a mode of RNA binding that is distinct from that of the well-characterized MS2 coat protein, suggesting new orthogonal systems for RNA labeling and purification.

Structural basis for the coevolution of a viral RNA–protein complex

The cocrystal structure of the PP7 bacteriophage coat protein in complex with its translational operator identifies a distinct mode of sequence-specific RNA recognition when compared to the well-characterized MS2 coat protein-RNA complex. The structure reveals the molecular basis of the PP7 coat protein's ability to selectively bind its cognate RNA, and it demonstrates that the conserved beta-sheet surface is a flexible architecture that can evolve to recognize diverse RNA hairpins.

Phage passage after extended processing in small‐virus‐retentive filters

Retention of a two small phages (PhiX-174 and pp7) by direct-flow small-virus-retentive filters [Viresolve NFP (normal-flow parvovirus), Virosart CPV (canine parvovirus), Ultipor DV20 and Planova 20N] was studied using a commercial-process fluid. Phage passage occurred in each filter type, particularly when overloaded with phage. Clearances of pp7 and PhiX-174 were similar for any given filter brand, arguing that the two phages are equivalent for testing small-virus-retentive filters. The patterns of flux under constant pressure and instantaneous LRV (log reduction value) in relationship to cumulative phage load differed between brands, consistent with the current industry understanding that each brand possesses specific performance attributes. Phages are a powerful and universal tool for evaluating filter performance. Validation of filter performance with phages such as pp7 or PhiX-174 as models for small mammalian viruses represents an attractive alternative to the current practice.

Molecular quantitative analysis of human viruses in California stormwater

Many human pathogenic viruses are transmitted via the oral–fecal route and water is one possible vector, representing a risk for public health. Sixty-one large-volume water samples from storm drains in California were processed by a two-step hollow fiber ultrafiltration procedure followed by molecular analysis for human enterovirus and adenovirus types. Each sample was spiked with a surrogate, the benign bacteriophage PP7. Both surrogate and human viruses were quantified by newly designed TaqMan ® PCR assays. Equations were developed that account for the main variables in the procedure: recovery of the ultrafiltration, efficiency of nucleic acid extraction, and effect of inhibitors on the amplification of viral targets. Adenovirus 40/41 was detected in one sample at 230 genomes per liter, and no other adenovirus or enterovirus types were found. Samples that resulted in nondetects are reported together with the corresponding sample-specific limit of detection ( S LOD), a useful tool when estimating the public health risk associated with the contact or ingestion of water. Virus concentrations did not correlate with traditional viable indicator concentrations or any of the physicochemical parameters measured. In contrast, coliform concentrations were correlated with total suspended solids. To our knowledge, this is the first study where all factors known to influence limits of detection have been investigated and integrated into equations that are widely applicable to the quantification of viruses or other microbial targets by PCR.

Validation of hollow fiber ultrafiltration and real-time PCR using bacteriophage PP7 as surrogate for the quantification of viruses from water samples

A quantitative real-time TaqMan ® PCR system for Pseudomonas aeruginosa bacteriophage PP7 was designed to detect PP7 as surrogate in performance tests of 2 hollow fiber ultrafiltration systems in series. Fifty-six storm water samples from 21 sites representing agricultural, urban and highway locations in California were collected. The optimized procedure gave recoveries of spiked PP7 of 64±4.8% (mean±SEM). The PP7 assay was validated over 5 orders of magnitude with an assay limit of detection of 5 gene copies per reaction volume. Sample-dependent variables like enzymatic inhibition during PCR analysis, filtration recovery and extraction efficiency were quantified and incorporated to calculate a specific sample limit of detection ( S LOD) for the spiked surrogate PP7. S LOD values were highly variable among samples; they were independent of physicochemical parameters including conductivity, turbidity, total suspended solids and pH but strongly correlated with the dilution factor required to relieve enzymatic inhibition during PCR analysis. To determine actual gene copies of PP7, a dilution approach was developed that involves assaying several dilutions within a range where inhibitors do not affect the efficiency of amplification and linear regression to determine the theoretical C t value when there is no inhibition. For the detection of viral pathogens, an internal standard like PP7 can be used to calculate filtration recoveries when quantifying pathogens and to determine whether filtration or inhibitor concentration affect nucleic acid extraction efficiency. Additionally, by defining S LOD values per sample and pathogenic organism analyzed, it should be possible to critically investigate the absence of detects for a particular pathogen and determine probabilities of risk associated with a specific sample limit of detection.

Stability and assembly in vitro of bacteriophage PP7 virus-like particles

BackgroundThe stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. The icosahedral capsid of the RNA bacteriophage PP7 is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes. This work determined the effects of these disulfides on the VLP's thermal stability.ResultsMeasurements of the thermal denaturation behavior of PP7 VLPs in the presence and absence of a reducing agent show that disulfide cross-links substantially stabilize them against thermal denaturation. Although dimers in the capsid are linked to one another by disulfides, the two subunits of dimers themselves are held together only by non-covalent interactions. In an effort to confer even greater stability a new cross-link was introduced by genetically fusing two coat protein monomers, thus producing a "single-chain dimer" that assembles normally into a completely cross-linked VLP. However, subunit fusion failed to increase the thermal stability of the particles, even though it stabilized the isolated dimer. As a step toward gaining control of the internal composition of the capsid, conditions that promote the assembly of PP7 coat protein dimers into virus-like particles in vitro were established.ConclusionThe presence of inter-dimer disulfide bonds greatly stabilizes the PP7 virus-like particle against thermal denaturation. Covalently cross-linking the subunits of the dimers themselves by genetically fusing them through a dipeptide linker sequence, offers no further stabilization of the VLP, although it does stabilize the dimer. PP7 capsids readily assemble in vitro in a reaction that requires RNA.

Cryo Electron Microscopy Reconstructions of the Leviviridae Unveil the Densest Icosahedral RNA Packing Possible

We solved the structures of the single-stranded RNA bacteriophages Qβ, PP7 and AP205 by cryo-electron microscopy. On the outside, the symmetrized electron density maps resemble the previously described cryo-electron microscopy structure of MS2. RNA density is present inside the capsids, suggesting that the genomic RNA of Qβ, PP7 and AP205, analogous to MS2, contains many coat protein-binding sites in addition to the hairpin on which assembly and packaging are initiated. All four bacteriophages harbour the same overall arrangement of the RNA, which is a unique combination of both triangles and pentagons. This combination has not been found in other icosahedral viruses, in which the RNA structures are either triangular or pentagonal. Strikingly, the unique RNA packing of the Leviviridae appears to deploy the most efficient method of RNA storage by obeying icosahedral symmetry.

Spatial heterogeneity and the stability of host-parasite coexistence

Spatially heterogeneous environments can theoretically promote more stable coexistence of hosts and parasites by reducing the risk of parasite attack either through providing permanent spatial refuges or through providing ephemeral refuges by reducing dispersal. In experimental populations of Pseudomonas aeruginosa and the bacteriophage PP7, spatial heterogeneity promoted stable coexistence of host and parasite, while coexistence was significantly less stable in the homogeneous environment. Phage populations were found to be persisting on subpopulations of sensitive bacteria. Transferring populations to fresh microcosms every 24 h prevented the development of permanent spatial refuges. However, the lower dispersal rates in the heterogeneous environment were found to reduce parasite transmission thereby creating ephemeral refuges from phage attack. These results suggest that spatial heterogeneity can stabilize an otherwise unstable host-parasite interaction even in the absence of permanent spatial refuges.

Comparison of 2 ultrafiltration systems for the concentration of seeded viruses from environmental waters

The use of ultrafiltration as a concentration method to recover viruses from environmental waters was investigated. Two ultrafiltration systems (hollow fiber and tangential flow) in a large- (100 L) and small-scale (2 L) configuration were able to recover greater than 50% of multiple viruses (bacteriophage PP7 and T1 and poliovirus type 2) from varying water turbidities (10-157 nephelometric turbidity units (NTU)) simultaneously. Mean recoveries (n = 3) in ground and surface water by the large-scale hollow fiber ultrafiltration system (100 L) were comparable to recoveries observed in the small-scale system (2 L). Recovery of seeded viruses in highly turbid waters from small-scale tangential flow (2 L) (screen and open channel) and hollow fiber ultrafilters (2 L) (small pilot) were greater than 70%. Clogging occurred in the hollow fiber pencil module and when particulate concentrations exceeded 1.6 g/L and 5.5 g/L (dry mass) in the screen and open channel filters, respectively. The small pilot module was able to filter all concentrates without clogging. The small pilot hollow fiber ultrafilter was used to test recovery of seeded viruses from surface waters from different geographical regions in 10-L volumes. Recoveries >70% were observed from all locations.

RNA recognition site of PP7 coat protein.

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.

Efficient and predictable recovery of viruses from water by small scale ultrafiltration systems

Current methods to concentrate viruses from large volumes of water are prone to inconsistent results and are costly and complex procedurally. Ultrafiltration can utilize size exclusion rather than adsorption and (or) elution to concentrate viruses and, therefore, may offer greater flexibility in developing methods that can provide more consistent recoveries among different viruses and widely varying water conditions. Two small scale ultrafiltration systems (hollow fiber and tangential flow) were tested with a virus suspended in 2 L of reagent grade, tap, ground, or surface water. Three model viruses were used (bacteriophages PP7 and T1 and poliovirus) to compare and characterize the recovery of viruses with the two ultrafiltration systems. Pretreatment of the ultrafilters with blocking agents and the use of elution agents can serve to prevent viral adsorption to the filter surface or to elute bound virus and keep viral agents suspended in the retentate. The use of a blocking and elution step concentrated viruses (>60% recovery) from widely varying water qualities, including surface water, such that a single method can be used to efficiently concentrate viruses from all of the water types tested. Both ultrafiltration systems appear to be able to efficiently recover viruses; however, the hollow fiber systems provided slightly better results in the 2-L volumes tested.

Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

The Three-Dimensional Structure of Bacteriophage PP7 from Pseudomonas aeruginosa at 3.7-Å Resolution

The three-dimensional structure of phage PP7 from Pseudomonas aeruginosa has been determined to 3.7-Å resolution. A comparison with distantly related small RNA phages showed that the biggest differences were found in the FG loops, forming the contacts around the fivefold and threefold axes. In contrast to the situation in other phages, the FG loops of phage PP7 are very similar in all three subunits. This supports the hypothesis that no switches are needed for the assembly control in these viruses. Some of the most conserved residues lie within the region involved in RNA binding in the related phages MS2 and seem to have the same function.

Structure determination of bacteriophage PP7 from Pseudomonas aeruginosa: from poor data to a good map.

The structure of bacteriophage PP7 from Pseudomomas aeruginosa was determined to 3.7 A resolution. Triclinic crystals of three forms were obtained, diffracting to between 4.5 and 3.4 A resolution. The quality of the crystals was exceptionally poor, leading to problems in the evaluation of the recorded images and to a final data set which would appear to be useless with standard criteria for protein crystals. In all crystal forms, the unit cell contains two icosahedral particles, providing 120-fold non-crystallographic symmetry. For two of the crystal forms, the particle orientations were calculated using the self-rotation function. The two particles in the asymmetric unit had very similar but distinct orientations. The position of the second particle was found using the Patterson function. Initial phases to 15 A resolution were calculated using the related phage MS2 as a model. Real-space averaging was performed and phases were extended from 15 A resolution to the limit of the data. The map was improved significantly by using only the 'high' resolution data in the resolution range 7-3.7 A, allowing the positions of most side chains to be determined. The better quality of the 7-3.7 A resolution map is presumably a consequence of the presence of satellite crystals. The position of the second particle was improved using the correlation coefficient in the averaging process to monitor the refinement by moving the particle around in small steps.