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Bacteriophage ϕX174 is a model virus for studies across the fields of structural biology, genetics, gut microbiomics, and synthetic biology, but did not have a high-resolution transcriptome until this work. In this study we used next-generation sequencing to measure the RNA produced from ϕX174 while infecting its host E. coli C. We broadly confirm the past transcriptome model while revealing several interesting deviations from previous knowledge. Additionally, we measure the strength of canonical ϕX174 promoters and terminators and discover both a putative new promoter that may be activated by heat shock sigma factors, as well as rediscover a controversial Rho-dependent terminator. We also provide evidence for the first antisense transcription observed in the Microviridae, identify two promoters that may be involved in generating this transcriptional activity, and discuss possible reasons why this RNA may be produced.

Most icosahedral viruses condense their genomes into volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded DNA (ssDNA) viruses. ssDNA genome packaging combines elements found in both double-stranded DNA (dsDNA) and ssRNA systems. Similar to dsDNA viruses, the genome is packaged into a preformed capsid. Like ssRNA viruses, there are numerous capsid-genome associations. In ssDNA microviruses, the DNA-binding protein J guides the genome between 60 icosahedrally ordered DNA binding pockets. It also partially neutralizes the DNA's negative phosphate backbone. ϕX174-related microviruses, such as G4 and α3, have J proteins that differ in length and charge organization. This suggests that interchanging J proteins could alter the path used to guide DNA in the capsid. Previously, a ϕXG4J chimera, in which the ϕX174 J gene was replaced with the G4 gene, was characterized. It displayed lethal packaging defects, which resulted in procapsids being removed from productive assembly. Here, we report the characterization of another inviable chimera, ϕXα3J. Unlike ϕXG4J, ϕXα3J efficiently packaged DNA but produced noninfectious particles. These particles displayed a reduced ability to attach to host cells, suggesting that internal DNA organization could distort the capsid's outer surface. Mutations that restored viability altered J-coat protein contact sites. These results provide evidence that the organization of ssDNA can affect both packaging and postpackaging phenomena. IMPORTANCE ssDNA viruses utilize icosahedrally ordered protein-nucleic acids interactions to guide and organize their genomes into preformed shells. As previously demonstrated, chaotic genome-capsid associations can inhibit ϕX174 packaging by destabilizing packaging complexes. However, the consequences of poorly organized genomes may extend beyond the packaging reaction. As demonstrated herein, it can lead to uninfectious packaged particles. Thus, ssDNA genomes should be considered an integral and structural virion component, affecting the properties of the entire particle, which includes the capsid's outer surface.

Bacteriophage plaque size measurement is essential for phage characterisation, but their manual size estimation requires a considerable amount of time and effort. In order to ease the work of phage researchers, we have developed an automated command-line application called Plaque Size Tool (PST) that can detect plaques of different morphology on the images of Petri dishes and measure plaque area and diameter. Plaque size measurements using PST showed no difference to those obtained with manual plaque size measurement in Fiji, indicating future results using PST are backwards compatible with prior measurements in the literature. PST can be applied to a range of lytic bacteriophages producing oval-shaped plaques, including bull’s-eye morphology. The application can also be used for titer calculation if most of the plaques are stand-alone. As laboratory automation becomes more commonplace, standardised and flexible open-source analytical tools like PST will be important parts of biofoundry and cloud lab bacteriophage workflows.

Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood. Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2. We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH). Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH ≥ 57%. Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media). Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival. Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live-attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion. We systematically recoded all nonoverlapping genes of the bacteriophage ΦX174 with codons rarely used in its Escherichia coli host. The fitness of recombinant viruses decreases as additional deoptimizing mutations are made to the genome, although not always linearly, and not consistently across genes. Combining deoptimizing mutations may reduce viral fitness more or less than expected from the effect size of the constituent mutations and we point out difficulties in untangling correlated compositional features. We test our model by optimizing the same genes and find that the relationship between codon usage and fitness does not hold for optimization, suggesting that wild-type ΦX174 is at a fitness optimum. This work highlights the need to better understand how selection acts on patterns of synonymous codon usage across the genome and provides a convenient system to investigate the genetic determinants of virulence.

In microviruses, 60 copies of the positively charged DNA binding protein J guide the single-stranded DNA genome into the icosahedral capsid. Consequently, ∼12% of the genome is icosahedrally ordered within virions. Although the internal volume of the ϕX174, G4, and α3 capsids are nearly identical, their genome lengths vary widely from 5,386 (ϕX174) to 6,067 (α3) nucleotides. As the genome size increases, the J protein’s length and charge decreases. The ϕX174 J protein is 37 amino acids long and has a charge of +12, whereas the 23-residue G4 and α3 proteins have respective +6 and +8 charges. While the large ϕX174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, ϕX174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (ϕXG4J) was generated by replacing the indigenous ϕX174 J gene with that of G4. The resulting mutant, ϕXG4J, was not viable on the level of plaque formation without ϕX174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than the wild type. Complementation-independent ϕXG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, ϕXG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene. IMPORTANCE Double-stranded DNA viruses typically package their genomes into a preformed capsid. In contrast, single-stranded RNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. Single-stranded DNA (ssDNA) viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling-circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential, and/or functionally undefined in-frame versions of A/A*-like proteins.

Viruses rely upon their hosts for biosynthesis of viral RNA, DNA and protein. This dependency frequently engenders strong selection for virus genome compatibility with potential hosts, appropriate gene regulation and expression necessary for a successful infection. While bioinformatic studies have shown strong correlations between codon usage in viral and host genomes, the selective factors by which this compatibility evolves remain a matter of conjecture. Engineered to include codons with a lesser usage and/or tRNA abundance within the host, three different attenuated strains of the bacterial virus ФX174 were created and propagated via serial transfers. Molecular sequence data indicate that biosynthetic compatibility was recovered rapidly. Extensive computational simulations were performed to assess the role of mutational biases as well as selection for translational efficiency in the engineered phage. Using bacteriophage as a model system, we can begin to unravel the evolutionary processes shaping codon compatibility between viruses and their host.

Here, we evaluated the removal of three representative human enteric viruses — adenovirus (AdV) type 40, coxsackievirus (CV) B5, and hepatitis A virus (HAV) IB — and one surrogate of human caliciviruses — murine norovirus (MNV) type 1 — by coagulation–rapid sand filtration, using water samples from eight water sources for 2 drinking water treatment plants in Japan. The removal ratios of a plant virus (pepper mild mottle virus; PMMoV) and two bacteriophages (MS2 and φX174) were compared with the removal ratios of human enteric viruses to assess the suitability of PMMoV, MS2, and φX174 as surrogates for human enteric viruses. The removal ratios of AdV, CV, HAV, and MNV, evaluated via the real-time polymerase chain reaction (PCR) method, were 0.8– 2.5-log10 when commercially available polyaluminum chloride (PACl, basicity 1.5) and virgin silica sand were used as the coagulant and filter medium, respectively. The type of coagulant affected the virus removal efficiency, but the age of silica sand used in the rapid sand filtration did not. Coagulation–rapid sand filtration with non-sulfated, high-basicity PACls (basicity 2.1 or 2.5) removed viruses more efficiently than the other aluminumbased coagulants. The removal ratios of MS2 were sometimes higher than those of the three human enteric viruses and MNV, whereas the removal ratios of φX174 tended to be smaller than those of the three human enteric viruses and MNV. In contrast, the removal ratios of PMMoV were similar to and strongly correlated with those of the three human enteric viruses and MNV. Thus, PMMoV appears to be a suitable surrogate for human enteric viruses for the assessment of the efficacy of coagulation–rapid sand filtration to remove viruses.

Canine brucellosis, caused by Brucella canis, is a persistent infectious reproductive disease in dogs. The absence of effective treatment to the intracellular pathogen and the irreversible consequence of infection makes the need of a specific vaccine urgent. Bacterial ghosts (BGs) are the empty envelopes of bacteria with no genome content inside, which emerge as a proper vaccine candidate due to its intact outer antigen. It is generally derived from a genetically engineered strain, through the expression of Bacteriophage phiX174 lysis E gene upon induction. In this study, we combined the homologous recombination (HR) and bacterial ghost technologies, generating a genetically stable B. canis ghost strain which bears no drug resistance gene. When the ghost strain grows to OD600 of 0.6, 100% inactivation can be achieved under 42°C in 60h. The resultant BGs showed guaranteed safety and comparable immunogenicity to a live vaccine. The bacterial B0419 protein was depleted during HR process, which is subsequently proved to work as a molecular tag in distinguishing natural infection and BGs immunization through ELISA. Additionally, the BGs also conferred protection against B. canis RM6/66 and B. melitensis 16M. Therefore, the application of current BGs as a vaccine candidate and the corresponding serological diagnostic approach may provide better B. canis prevention strategy.

In order to assure the microbial safety of drinking water, UVC-LED treatment has emerged as a possible technology to replace the use of conventional low pressure (LP) mercury vapor UV lamps. In this investigation, inactivation of Human Enteric Virus (HuEV) surrogates with UVC-LEDs was investigated in a water disinfection system, and kinetic model equations were applied to depict the surviving infectivities of the viruses. MS2, Qβ, and ΦX 174 bacteriophages were inoculated into sterile distilled water (DW) and irradiated with UVC-LED printed circuit boards (PCBs) (266nm and 279nm) or conventional LP lamps. Infectivities of bacteriophages were effectively reduced by up to 7-log after 9mJ/cm2 treatment for MS2 and Qβ, and 1mJ/cm2 for ΦX 174. UVC-LEDs showed a superior viral inactivation effect compared to conventional LP lamps at the same dose (1mJ/cm2). Non-log linear plot patterns were observed, so that Weibull, Biphasic, Log linear-tail, and Weibull-tail model equations were used to fit the virus survival curves. For MS2 and Qβ, Weibull and Biphasic models fit well with R2 values approximately equal to 0.97-0.99, and the Weibull-tail equation accurately described survival of ΦX 174. The level of UV-susceptibility among coliphages measured by the inactivation rate constant, k, was statistically different (ΦX 174 (ssDNA)>MS2, Qβ (ssRNA)), and indicated that sensitivity to UV was attributed to viral genetic material.

Although microviruses do not possess a visible tail structure, one vertex rearranges after interacting with host lipopolysaccharides. Most examinations of host range, eclipse, and penetration were conducted before this “host-induced” unique vertex was discovered and before DNA sequencing became routine. Consequently, structure-function relationships dictating host range remain undefined. Biochemical and genetic analyses were conducted with two closely related microviruses, α3 and ST-1. Despite ∼90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. Virions attached and eclipsed to both native and unsusceptible hosts; however, they breached only the native host’s cell wall. This suggests that unsusceptible host-phage interactions promote off-pathway reactions that can inactivate viruses without penetration. This phenomenon may have broader ecological implications. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: genetically mutant progeny from coinfected cells did not display the phenotype. Thus, mutant isolation required populations generated in environments with low multiplicities of infection (MOI), a phenomenon that may have impacted past host range studies in both prokaryotic and eukaryotic systems. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid. IMPORTANCE To expand host range, viruses must interact with unsusceptible host cell surfaces, which could be detrimental. As observed in this study, virions were inactivated without genome penetration. This may be advantageous to potential new hosts, culling the viral population from which an expanded host range mutant could emerge. When identified, altered host range mutations were recessive. Accordingly, isolation required populations generated in low-MOI environments. However, in laboratory settings, viral propagation includes high-MOI conditions. Typically, infected cultures incubate until all cells produce progeny. Thus, coinfections dominate later replication cycles, masking recessive host range expansion phenotypes. This may have impacted similar studies with other viruses. Last, structural and genetic data could be used to predict site-directed mutant phenotypes, which may broaden the classic antireceptor definition to include interfaces between capsid complexes.

To assess how B cell phenotype analysis correlates with antigen responses in patients with class switch recombination defects (CSRD) we quantified memory B cells by flow-cytometry and immunized CSRD patients with the neoantigen bacteriophage phiX174 (phage). CSRD patients showed uniformly absent or markedly reduced switched memory B cells (IgM-IgD-CD27+). CD40L patients had reduced CD27+ memory B cells (both non-switched and switched). In NEMO patients, results varied depending on the IKKγ gene variant. Three of four AID patients had normal percentages of CD27+ memory B cells while CD27+IgM-IgD- switched memory B cells were markedly reduced in all AID patients. Antibody response to phage was remarkably decreased with lack of memory amplification and class-switching in immunized CD40L, UNG deficient, and NEMO patients. Distinct B-cell phenotype pattern correlated with abnormal antibody responses to a T-cell dependent neoantigen, representing a powerful tool to identify CSRD patients.

Waterborne viruses are increasingly being considered in risk assessment schemes. In general, virus detection by culture methods is time consuming. In contrast, detection by quantitative polymerase chain reaction (qPCR) is more rapid and therefore, more suitable for monitoring. At present, qPCR lacks the essential ability for discriminating between infectious and non-infectious viruses, thus limiting its applicability for monitoring disinfection processes. In this study, a method was developed to quantify UV inactivation by long amplicon (LA)-qPCR. Bacteriophage phiX174 was used as a surrogate for human pathogenic viruses. A qPCR protocol was developed with new sets of primers, resulting in amplicon lengths of 108, 250, 456, 568, 955, 1063, 1544, and 1764 nucleotides. The log reduction of gene copies increased with increasing amplicon length. Additional treatment with the intercalating dye, PMA, had no effect, indicating that the bacteriophage capsids were not damaged by low pressure UV irradiation. A qPCR of nearly the complete genome (approx. 5000 nucleotides) showed similar results to the plaque assay. The log reduction in qPCR correlates with [specific amplicon length x UV dose]. The normalized DNA effect constant can be applied to calculate phiX174 inactivation based on qPCR detection.

Researchers in evolutionary genetics recently have recognized an exciting opportunity in decomposing beneficial mutations into their proximal, mechanistic determinants. The application of methods and concepts from molecular biology and life history theory to studies of lytic bacteriophages (phages) has allowed them to understand how natural selection sees mutations influencing life history. This work motivated the research presented here, in which we explored whether, under consistent experimental conditions, small differences in the genome of bacteriophage φX174 could lead to altered life history phenotypes among a panel of eight genetically distinct clones. We assessed the clones’ phenotypes by applying a novel statistical framework to the results of a serially sampled parallel infection assay, in which we simultaneously inoculated each of a large number of replicate host volumes with ∼1 phage particle. We sequentially plated the volumes over the course of infection and counted the plaques that formed after incubation. These counts served as a proxy for the number of phage particles in a single volume as a function of time. From repeated assays, we inferred significant, genetically determined heterogeneity in lysis time and burst size, including lysis time variance. These findings are interesting in light of the genetic and phenotypic constraints on the single-protein lysis mechanism of φX174. We speculate briefly on the mechanisms underlying our results, and we discuss the potential importance of lysis time variance in viral evolution.

Infectious airborne microbes, including many pathological microbes that cause respiratory infections, are commonly found in medical facilities and constitute a serious threat to human health. Thus, an effective method for reducing the number of microbes floating in the air will aid in the minimization of the incidence of respiratory infectious diseases. Here, we demonstrate that chlorine dioxide (ClO2) gas at extremely low concentrations, which has no detrimental effects on human health, elicits a strong effect to inactivate bacteria and viruses and significantly reduces the number of viable airborne microbes in a hospital operating room. In one set of experiments, a suspension of Staphylococcus aureus, bacteriophage MS2, and bacteriophage ΦX174 were released into an exposure chamber. When ClO2 gas at 0.01 or 0.02 parts per million (ppm, volume/volume) was present in the chamber, the numbers of surviving microbes in the air were markedly reduced after 120 min. The reductions were markedly greater than the natural reductions of the microbes in the chamber. In another experiment, the numbers of viable airborne bacteria in the operating room of a hospital collected over a 24-hour period in the presence or absence of 0.03 ppm ClO2 gas were found to be 10.9 ± 6.7 and 66.8 ± 31.2 colony-forming units/m3 (n = 9, p < 0.001), respectively. Taken together, we conclude that ClO2 gas at extremely low concentrations (≤0.03 ppm) can reduce the number of viable microbes floating in the air in a room. These results strongly support the potential use of ClO2 gas at a non-toxic level to reduce infections caused by the inhalation of pathogenic microbes in nursing homes and medical facilities.

Bacteriophage ϕX174 uses a decamer of DNA piloting proteins to penetrate its host. These proteins oligomerize into a cell wall-spanning tube, wide enough for genome passage. While the inner surface of the tube is primarily lined with inward-facing amino acid side chains containing amide and guanidinium groups, there is a 28 &Aring, long section near the tube&rsquo, s C-terminus that does not exhibit this motif. The majority of the inward-facing residues in this region are conserved across the three ϕX174-like clades, suggesting that they play an important role during genome delivery. To test this hypothesis, and explore the general function of the tube&rsquo, s inner surface, non-glutamine residues within this region were mutated to glutamine, while existing glutamine residues were changed to serine. Four of the resulting mutants had temperature-dependent phenotypes. Virion assembly, host attachment, and virion eclipse, defined as the cell&rsquo, s ability to inactivate the virus, were not affected. Genome delivery, however, was inhibited. The results support a model in which a balance of forces governs genome delivery: potential energy provided by the densely packaged viral genome and/or an osmotic gradient move the genome into the cell, while the tube&rsquo, s inward facing glutamine residues exert a frictional force, or drag, that controls genome release.

Aeromonas veronii is a conditional pathogen causing high mortality in many freshwater fish species worldwide. Bacterial ghosts are nonliving Gram-negative bacteria devoid of cytoplasmic contents, which induce protective immunity against microbial pathogens. The aims of this study were: a) to produce A. veronii ghost (AVG) constructed by PhiX174 gene E; b) to evaluate the specific, non-specific immune effects and protective immunity of AVG against A. veronii in koi. The lysis plasmid pBBR-E was constructed by cloning PhiX174 gene E into the broad-host-range vector pBBR1MCS2, and then transformed into A. veronii 7231. AVG was generated by increasing the incubation temperature up to 42 °C. Lysis of A. veronii occurred 3 h after temperature induction and completed in 12 h. The efficiency of ghost induction was 99.9998 ± 0.0002%. Koi were immunized intraperitoneally with AVG, formalin-killed bacteria (FKC) or phosphate buffered saline (PBS) respectively, and then respiratory burst (RB), myeloperoxidase (MPO), lysozyme (LZM), malondialdehyde (MDA), complement 3 (C3) and antibody activities were examined in serum. Compared with negative control of PBS, the RB, MPO, LZM activities were significantly higher in koi immunized with AVG (P 0.05). Nevertheless, the MDA activities of AVG treatment were significantly lower than those of PBS treatment (P 0.05). The serum agglutination titers and IgM antibody titers in AVG group were significantly higher than those in FKC or PBS groups. After challenged with the parent strain A. veronii 7231, the average mortality of AVG group was significantly lower than that of FKC and PBS groups (P 0.05) and the relative percent survival (RPS) of AVG group (73.92%) was higher than that of FKC group (43.48%). Therefore, AVG have the potential to induce protective immunity and they may be ideal vaccine candidates against A. veronii in koi.

Two scaffolding proteins orchestrate ϕX174 morphogenesis. The internal scaffolding protein B mediates the formation of pentameric assembly intermediates, whereas the external scaffolding protein D organizes 12 of these intermediates into procapsids. Aromatic amino acid side chains mediate most coat-internal scaffolding protein interactions. One residue in the internal scaffolding protein and three in the coat protein constitute the core of the B protein binding cleft. The three coat gene codons were randomized separately to ascertain the chemical requirements of the encoded amino acids and the morphogenetic consequences of mutation. The resulting mutants exhibited a wide range of recessive phenotypes, which could generally be explained within a structural context. Mutants with phenylalanine, tyrosine, and methionine substitutions were phenotypically indistinguishable from the wild type. However, tryptophan substitutions were detrimental at two sites. Charged residues were poorly tolerated, conferring extreme temperature-sensitive and lethal phenotypes. Eighteen lethal and conditional lethal mutants were genetically and biochemically characterized. The primary defect associated with the missense substitutions ranged from inefficient internal scaffolding protein B binding to faulty procapsid elongation reactions mediated by external scaffolding protein D. Elevating B protein concentrations above wild-type levels via exogenous, cloned-gene expression compensated for inefficient B protein binding, as did suppressing mutations within gene B. Similarly, elevating D protein concentrations above wild-type levels or compensatory mutations within gene D suppressed faulty elongation. Some of the parental mutations were pleiotropic, affecting multiple morphogenetic reactions. This progressively reduced the flux of intermediates through the pathway. Accordingly, multiple mechanisms, which may be unrelated, could restore viability. IMPORTANCE Genetic analyses have been instrumental in deciphering the temporal events of many biochemical pathways. However, pleiotropic effects can complicate analyses. Vis-à-vis virion morphogenesis, an improper protein-protein interaction within an early assembly intermediate can influence the efficiency of all subsequent reactions. Consequently, the flux of assembly intermediates cumulatively decreases as the pathway progresses. During morphogenesis, ϕX174 coat protein participates in at least four well-defined reactions, each one characterized by an interaction with a scaffolding or structural protein. In this study, genetic analyses, biochemical characterizations, and physiological assays, i.e., elevating the protein levels with which the coat protein interacts, were used to elucidate pleiotropic effects that may alter the flux of intermediates through a morphogenetic pathway.

Although ϕX174 DNA pilot protein H is monomeric during procapsid assembly, it forms an oligomeric tube on the host cell surface. Reminiscent of a double-stranded DNA phage tail in form and function, the H tube transports the single-stranded ϕX174 genome across the Escherichia coli cell wall. The 2.4-Å resolution H-tube crystal structure suggests functional and energetic mechanisms that may be common features of DNA transport through virally encoded conduits.

The inactivation effects of pressurized CO2 against bacteriophage Qβ and ΦX174 were investigated under the pressure of 0.3-0.9 MPa, initial concentration of 10(7)-10(9) PFU/mL, and temperature of 17.8°C-27.2°C. The optimum conditions were found to be 0.7 MPa and an exposure time of 25 min. Under identical treatment conditions, a greater than 3.3-log reduction in bacteriophage Qβ was achieved by CO2, while a nearly 3.0 log reduction was observed for phage ΦX174. The viricidal effects of N2O (an inactivation gas with similar characteristics to CO2), normal acid (HCl), and CO2 treatment with phosphate buffered saline affirmed the chemical nature of CO2 treatment. The pumping cycle, depressurization rate, and release of intracellular substances caused by CO2 were its viricidal mechanisms. The results indicate that CO2 has the potential for use as a disinfectant without forming disinfection by-products.

The øX174 DNA pilot protein H forms an oligomeric DNA-translocating tube during penetration. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive ( cs ) and temperature-sensitive ( ts ) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

With a growing world population, the lack of reliable water sources is becoming an increasing problem. Reusing greywater could alleviate this problem. When reusing greywater for crop irrigation it is paramount to ensure the removal of pathogenic organisms. This study compared the pathogen removal efficiency of pine bark and activated charcoal filters with that of conventional sand filters at three organic loading rates. The removal efficiency of Escherichia coli O157:H7 decreased drastically when the organic loading rate increased fivefold in the charcoal and sand filters, but increased by 2 log10 in the bark filters. The reduction in the virus model organism coliphage phiX174 remained unchanged with increasing organic loading in the charcoal and sand filters, but increased by 2 log10 in the bark filters. Thus, bark was demonstrated to be the most promising material for greywater treatment in terms of pathogen removal.

The 5386 nucleotide bacteriophage øX174 genome has a complicated architecture that encodes 11 gene products via overlapping protein coding sequences spanning multiple reading frames. We designed a 6302 nucleotide synthetic surrogate, øX174.1, that fully separates all primary phage protein coding sequences along with cognate translation control elements. To specify øX174.1f, a decompressed genome the same length as wild type, we truncated the gene F coding sequence. We synthesized DNA encoding fragments of øX174.1f and used a combination of in vitro- and yeast-based assembly to produce yeast vectors encoding natural or designer bacteriophage genomes. We isolated clonal preparations of yeast plasmid DNA and transfected E. coli C strains. We recovered viable øX174 particles containing the øX174.1f genome from E. coli C strains that independently express full-length gene F. We expect that yeast can serve as a genomic ‘drydock’ within which to maintain and manipulate clonal lineages of other obligate lytic phage.

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro . In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo . We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.

In an earlier study, protein-based barriers to horizontal gene transfer were investigated by placing the bacteriophage G4 G gene, encoding the major spike protein, into the φX174 genome. The foreign G protein promoted off-pathway assembly reactions, resulting in a lethal phenotype. After three targeted genetic selections, one of two foreign spike proteins was productively integrated into the φX174 system: the complete G4 or a recombinant G4/φX174 protein (94% G4:6% φX174). However, strain fitness was very low. In this study, the chimeras were characterized and experimentally evolved. Inefficient assembly was the primary contributor to low fitness: accordingly, mutations affecting assembly restored fitness. The spike protein preference of the ancestral and evolved strains was determined in competition experiments between the foreign and φX174G proteins. Before adaptation, both G proteins were incorporated into virions; afterwards, the foreign proteins were strongly preferred. Thus, a previously inhibitory protein became the preferred substrate during assembly.

Although the ϕX174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wild-type length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein. IMPORTANCE The H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA-translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the α-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long α-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using in-frame insertions and deletions as presented herein.

Sunlight is known to be a pertinent factor governing the infectivity of waterborne viruses in the environment. Sunlight inactivates viruses via endogenous inactivation (promoted by absorption of solar light in the UVB range by the virus) and exogenous processes (promoted by adsorption of sunlight by external chromophores, which subsequently generate inactivating reactive species). The extent of inactivation is still difficult to predict, as it depends on multiple parameters including virus characteristics, solution composition, season and geographical location. In this work, we adapted a model typically used to estimate the photodegradation of organic pollutants, APEX, to explore the fate of two commonly used surrogates of human viruses (coliphages MS2 and ϕX174) in waste stabilization pond and natural surface water. Based on experimental data obtained in previous work, we modeled virus inactivation as a function of water depth and composition, as well as season and latitude, and we apportioned the contributions of the different inactivation processes to total inactivation. Model results showed that ϕX174 is inactivated more readily than MS2, except at latitudes >60°. ϕX174 inactivation varies greatly with both season (20-fold) and latitude (10-fold between 0 and 60°), and is dominated by endogenous inactivation under all solution conditions considered. In contrast, exogenous processes contribute significantly to MS2 inactivation. Because exogenous inactivation can be promoted by longer wavelengths, which are less affected by changes in season and latitude, MS2 exhibits smaller fluctuations in inactivation throughout the year (10-fold) and across the globe (3-fold between 0 and 60°) compared to ϕX174. While a full model validation is currently not possible due to the lack of sufficient field data, our estimated inactivation rates corresponded well to those reported in field studies. Overall, this study constitutes a step toward estimating microbial water quality as a function of spatio-temporal information and easy-to-determine solution parameters.

It has been well established that chemical mutagenesis has adverse fitness effects in RNA viruses, often leading to population extinction. This is mainly a consequence of the high RNA virus spontaneous mutation rates, which situate them close to the extinction threshold. Single-stranded DNA viruses are the fastest-mutating DNA-based systems, with per-nucleotide mutation rates close to those of some RNA viruses, but chemical mutagenesis has been much less studied in this type of viruses. Here, we serially passaged bacteriophage ϕX174 in the presence of the nucleoside analogue 5-fluorouracil (5-FU). We found that 5-FU was unable to trigger population extinction for the range of concentrations tested, but it negatively affected viral adaptability. The phage evolved partial drug resistance, and parallel nucleotide substitutions appearing in independently evolved lines were identified as candidate resistance mutations. Using site-directed mutagenesis, two single-nucleotide substitutions in the lysis protein E (T572C and A781G) were shown to be selectively advantageous in the presence of 5-FU. In RNA viruses, base analogue resistance is often mediated by changes in the viral polymerase, but this mechanism is not possible for ϕX174 and other single-stranded DNA viruses because they do not encode their own polymerase. In addition to increasing mutation rates, 5-FU produces a wide variety of cytotoxic effects at the levels of replication, transcription, and translation. We found that substitutions T572C and A781G lost their ability to confer 5-FU resistance after cells were supplemented with deoxythymidine, suggesting that their mechanism of action is at the DNA level. We hypothesize that regulation of lysis time may allow the virus to optimize progeny size in cells showing defects in DNA synthesis.

Bacterial ghost is a novel vaccine platform, and its safe and efficient production depends largely upon a suitable and functional vector. In this study, a series of temperature-inducible plasmids, carrying Phix174 lysis gene E and/or staphylococcal nuclease A (SNA) gene, were constructed and evaluated in Escherichia coli. The results showed that the direct product of SNA (pBV220-SNA) could degrade the plasmid and genomic DNA of E. coli while the fusion product of gene E and partial Cro gene (pKF396M-2) lost the ability to lyse the host strain. The insertion of enhancer T7g10 elements and Shine-Dalgarno box (ESD) between them (pKF396M-3) could resume the function of gene E. Using plasmid pKF396M-4 with gene E and SNA, respectively, under the immediate control of promoter pR and pL, the remnant plasmids and genomic DNA of E. coli were eliminated, and the rates of inactivation increased by two orders of magnitude over that obtained with the exclusive use of E-mediated lysis plasmid. By substituting these two genes with customized multiple cloning sites sequences, the plasmid could be modified to a dual expression vector (pKF396M-5).

GABA tea is a tea product that contains a high level of γ-aminobutyric acid (GABA). Previous study has demonstrated a synergistic effect of GABA tea and copper ions on DNA breakage. This study further explored whether zinc (Zn), a nonredox metal, modulated DNA cleavage induced by GABA tea extract. In a cell-free system, Zn(2+) significantly enhanced GABA tea extract and (-)-epigallocatechin-3-gallate (EGCG)- or H(2)O(2)-induced DNA damage at 24 h of incubation. Additionally, low dosages of GABA tea extract (1-10 μg/mL) possessed pro-oxidant activity to increase H(2)O(2)/Zn(2+)-induced DNA cleavage in a dose-dependent profile. By use of various reactive oxygen scavengers, it was observed that glutathione, catalase, and potassium iodide effectively inhibited DNA degradation caused by the GABA tea extract/H(2)O(2)/Zn(2+) system. Moreover, the data showed that the GABA tea extract itself (0.5-5 mg/mL) could induce DNA cleavage in a long-term exposure (48 h). EGCG, but not the GABA tea extract, enhanced H(2)O(2)-induced DNA cleavage. In contrast, GABA decreased H(2)O(2)- and EGCG-induced DNA cleavage, suggesting that GABA might contribute the major effect on the antioxidant activity of GABA tea extract. Furthermore, a comet assay revealed that GABA tea extract (0.25 mg/mL) and GABA had antioxidant activity on H(2)O(2)-induced DNA breakage in human peripheral lymphocytes. Taken together, these findings indicate that GABA tea has the potential of both pro-oxidant and antioxidant. It is proposed that a balance between EGCG-induced pro-oxidation and GABA-mediated antioxidation may occur in a complex mixture of GABA tea extract.

The photolytic and photocatalytic disinfection of three bacteriophages (MS2, ΦX174 and PR772) under both artificial UV irradiation and natural sunlight was studied. A large variation was seen in the relative responses of phages to the two light sources, while solar photocatalysis showed promising disinfection capabilities for all three phages. Under artificial UV, the resistance of phages to both photolytic and artificial treatment was found to decrease in the following order: PR772>ΦX174>MS2. Indeed, 3-log inactivation of PR772 was not achieved after 25 h of illumination in either the absence or presence of catalyst. Under solar treatment, PR772 was inactivated far more readily, 3-log inactivation being achieved within an hour in the presence of catalyst in two trials. It is thought that the variation in solar intensity during the trials had a major effect on disinfection rates, as kinetics for phages varied considerably under different weather conditions.

Human urine has the potential to be a sustainable, locally and continuously available source of nutrients for agriculture. Phosphate can be efficiently recovered from human urine in the form of the mineral struvite (MgNH4PO4·6H2O). However, struvite formation may be coupled with the precipitation of other constituents present in urine including pathogens, pharmaceuticals, and heavy metals. To determine if struvite fertilizer presents a microbiological health risk to producers and end users, we characterized the fate of a human virus surrogate (phage ΦX174) and the eggs of the helminth Ascaris suum during a low-cost struvite recovery process. While the concentration of phages was similar in both the struvite and the urine, Ascaris eggs accumulated within the solid during the precipitation and filtration process. Subsequent air-drying of the struvite filter cake partially inactivated both microorganisms; however, viable Ascaris eggs and infective phages were still detected after several days of drying. The infectivity of both viruses and eggs was affected by the specific struvite drying conditions: higher inactivation generally occurred with increased air temperature and decreased relative humidity. On a log-log scale, phage inactivation increased linearly with decreasing moisture content of the struvite, while Ascaris inactivation occurred only after achieving a minimum moisture threshold. Sunlight exposure did not directly affect the infectivity of phages or Ascaris eggs in struvite cakes, though the resultant rise in temperature accelerated the drying of the struvite cake, which contributed to inactivation.

Endonucleases that generate double-strand breaks in DNA often possess two identical subunits related by rotational symmetry, arranged so that the active sites from each subunit act on opposite DNA strands. In contrast to many endonucleases, Type IIP restriction enzyme BcnI, which recognizes the pseudopalindromic sequence 5'-CCSGG-3' (where S stands for C or G) and cuts both DNA strands after the second C, is a monomer and possesses a single catalytic center. We show here that to generate a double-strand break BcnI nicks one DNA strand, switches its orientation on DNA to match the polarity of the second strand and then cuts the phosphodiester bond on the second DNA strand. Surprisingly, we find that an enzyme flip required for the second DNA strand cleavage occurs without an excursion into bulk solution, as the same BcnI molecule acts processively on both DNA strands. We provide evidence that after cleavage of the first DNA strand, BcnI remains associated with the nicked intermediate and relocates to the opposite strand by a short range diffusive hopping on DNA.

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks φX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.

Bacterial ghosts (BGs) are empty bacterial envelopes generated by expulsion of the bacterial genome and cytoplasmic contents from bacterial cells, and the process is mediated by lysis protein E encoded on bacteriophage PhiX174. BGs represent a new approach in vaccine development and have been applied to a variety of gram-negative bacterial vaccine candidates. In this study, a BG vaccine generated from Salmonella enteritidis ( S. enteritidis ) strain DH091 was prepared using the highly efficient plasmid, pBV-mE. The efficacy of the BG vaccine was tested using 75 chicks (Gallus gallus) kept under specific pathogen-free (SPF) conditions. A comprehensive evaluation of the immune response, including humoral and cellular immune responses, interferon-γ (IFN-γ) and interleukin-4 (IL-4) production, and histopathology of various tissues, was performed in BG-vaccinated animals subsequently challenged with S. enteritidis . The results were compared with animals that were immunized with the inactivated vaccine. S. enteritidis ghosts not only promoted the generation of high titer antibodies and IFN-γ and IL-4 production but also stimulated a significant increase in CD8 + and CD4 + T lymphocytes. In particular, the dramatic increase in CD8 + T cells indicated that the vaccine was able to induce clearance of intracellular Salmonella . The protective effects of BG vaccination in SPF chicks against 5 × 10 9 colony forming units of S. enteritidis were a result of the induction of a more effective immune response than that observed with the inactivated vaccine. These findings demonstrate the potential of S. enteritidis ghosts to be used as effective vaccines.

To determine bacteriophage PhiX174's ecological niche, 783 Escherichia coli isolates were screened for susceptibility. Sensitive strains are diverse regarding their phylogenies and core lipopolysaccharides (LPS), but all have rough phenotypes. Further analysis of E. coli K-12 LPS mutants revealed that PhiX174 can use a wide diversity of LPS structures to initiate its infectious process.

The ability for a virus to expand its host range is dependent upon a successful mode of viral entry. As such, the host range of the well-studied ΦX174 bacteriophage is dictated by the presence of a particular lipopolysaccharide (LPS) on the bacterial surface. The mutant ΦX174 strain JACS-K, unlike its ancestor, is capable of infecting both its native host Escherichia coli C and E. coli K-12, which does not have the necessary LPS. The conversion of an alanine to a very reactive threonine on its virion surface was found to be responsible for the strain's expanded host range.

An improved silver-ammonia staining method for DNA on polyacrylamide gels is described. In this method, staining of DNA using silver-ammonia complex allows high sensitivity, low cost, low toxicity, and simple protocol without requiring fixation and sensitization steps. The protocol takes less than 40 min to complete, with a detection limit of 1.5 pg of single DNA band on polyacrylamide gels, approximately 30-fold higher than that of original silver-ammonia staining method. Furthermore, this novel technique not only exhibits high sensitivity for large DNA fragment, but also shows a better trend to detect low-base-pair DNA compared with other silver staining methods.

Atomic force microscopy (AFM) was used to study the attachment of phiX174, MS2, and Aichi viruses on sands of different surface properties: oxide-removed (clean), goethite-coated, and aluminum oxide-coated. Interaction forces between viruses and sand surfaces were measured by contact mode AFM using tips coated with particles of each virus. Column experiments were conducted to quantify the macroscopic transport and retention of the viruses in sand. The average adhesion force measured with AFM was highest between aluminum oxide-coated sand and all three viruses, followed by goethite-coated sand, and was significantly lower on oxide-removed sand. Among the viruses, adhesion on goethite-coated and aluminum oxide-coated sands followed the order of MS2AichiphiX174, and on oxide-removed sand it was phiX174AichiMS2. Column breakthrough results revealed the same retention trend, which was completely consistent with AFM force measurements. Strong electrostatic attraction and, to a lesser extent, hydrophobic interactions are responsible for the much greater removal of all three viruses observed in the oxide-coated sands compared to the oxide-removed sand. Mass recovery data indicate that the removal of phiX174, MS2, and Aichi was largely reversible when eluted with 3% beef extract solution at pH 9.5. The Derjaguin-Landau-Verwey-Overbeek (DLVO) and extended DLVO theories provided correct qualitative predictions on the deposition trend observed in the experiments. This study, to the best of our knowledge, was the first to employ AFM to directly measure interaction forces between viruses and solid surfaces; and it was the first to evaluate the retention and transport behavior of Aichi virus, a human pathogen.

Among 40 Escherichia coli urine isolates from renal transplant recipients (Galveston, TX, 2003 to 2005), sequence type ST131 (O25:H4) was highly prevalent (representing 35% of isolates overall and 60% of fluoroquinolone-resistant isolates), virulent appearing, antimicrobial resistant (but extended-spectrum-cephalosporin susceptible), and associated with black race. Pulsotypes were diverse; some were linked to other locales. ST131 emerged significantly during the study period. These findings suggest that E. coli ST131 may constitute an important new multidrug-resistant threat to renal transplant recipients.

Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.

The first α-helices of Microviridae external scaffolding proteins function as coat protein substrate specificity domains. Mutations in this helix can lengthen the lag phase before progeny production. 5′ deletion genes, encoding N-terminal deletion proteins, were constructed on plasmids and in the oX174 genome. Proteins lacking the first seven amino acids were able to rescue a nullD mutant when expressed from a plasmid. However, the lag phase before progeny production was lengthened. The oX174 mutant with the corresponding genomic gene grew very poorly. The molecular basis of the defective phenotype was complex. External scaffolding protein levels were reduced compared to wild-type and most of the viral coat protein in mutant infected cells appears to be siphoned off the assembly pathway. Second-site suppressors of the growth defects were isolated and appear to act via two different mechanisms. One class of suppressors most likely acts by altering mutant external scaffolding protein expression while the second class of suppressors appears to act on the level of protein–protein interactions.

Background It is unknown whether a prolonged period of bed rest will affect human immune responses, particularly in female subjects. Objective We sought to measure immune responses in adult female subjects exposed to prolonged bed rest. Methods Adult (25-40 years) female volunteers (n = 24) were maintained in a supine (6° tilt) head-down bed-rest (HDBR) position for 60 days: 8 with HDBR only, 8 with HDBR and regular muscular exercise, and 8 with HDBR and dietary protein supplementation. Subjects were immunized with bacteriophage φX-174. Antibody production and plasma cytokine levels were determined. Results The rate of primary antibody production of the HDBR plus exercise group increased faster ( P = .01) and to a higher level versus that of the HDBR-only group ( P = .03) and that of the HDBR plus diet group (trend P = .08). The rates of secondary antibody production between the 3 groups were similar, but the level of antibody in the HDBR plus exercise group remained higher versus that in the HDBR-only group ( P = .03). Both the HDBR ( P = .001) and HDBR plus diet ( P = .02) groups had time-related progressive increases in TNF-α receptor levels, but the HDBR plus exercise group remained at baseline. The HDBR plus exercise group experienced an acute increase in IL-1 receptor antagonist levels versus the HDBR ( P = .02) and the HDBR plus diet ( P = .02) groups, with similar increases in RANTES levels. Conclusions The exercise countermeasure accelerated primary antibody production and increased antibody levels to bacteriophage φX-174 and also opposed the potentially harmful effects of increased TNF-α levels caused by prolonged bed rest, possibly by activating the anti-inflammatory cytokine IL-1 receptor antagonist and the chemotactic factor RANTES.

Bacterial ghosts may be generated by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The Edwardsiella tarda ghost (ETG) vaccine was prepared using this technology and tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+ T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease.

We report detailed genetic and immunologic studies in a patient diagnosed with adenosine deaminase (ADA) deficiency and combined immune deficiency at age 5 years. At the time of diagnosis, although all other lymphocyte subsets were depleted, circulating CD8(+) T cells with a terminally differentiated phenotype were abundant and expressed normal ADA activity due to a reversion mutation in a CD8(+) T cell or precursor. Over the first 9 months of replacement therapy with PEG-ADA, the patient steadily accumulated mature naïve CD4(+) and CD8(+) T cells, as well as CD4(+)/FOXP3(+) regulatory T cells, consistent with restoration of a functional cellular immune system. While CD19(+) naïve B cells also accumulated in response to PEG-ADA therapy, a high proportion of these B cells exhibited an immature surface marker phenotype even after 9 months, and immunization with neoantigen bacteriophage varphiX174 demonstrated a markedly subnormal humoral immune response. Our observations in this single patient have important implications for gene therapy of human ADA deficiency, as they indicate that ADA expression within even a large circulating lymphocyte population may not be sufficient to support adequate immune reconstitution. They also suggest that an immature surface marker phenotype of the peripheral B cell compartment may be a useful surrogate marker for incomplete humoral immune reconstitution during enzyme replacement, and possibly other forms of hematopoietic cell therapies.

Genetic variation in viral structural proteins is often explained by evolutionary escape of strong host defenses through processes such as immune evasion, host switching, and tissue tropism. An understanding of the mechanisms driving evolutionary change in virus surface proteins is key to designing effective intervention strategies to disease emergence. This study investigated the predictability of virus genomic evolution in response to highly specific differences in host receptor structure. The bacteriophage ΦX174 was evolved on three E. coli mutant hosts, each differing only by a single sugar group in the lipopolysaccharides, used for phage attachment. Large phage populations were used in order to maximize the amount of sequence space explored by mutation, and thus the potential for parallel evolution. Repeatability was assessed by genome sequencing of multiple isolates from endpoint populations and by fitness of the endpoint population relative to its ancestor. Evolutionary lines showed similar magnitudes of fitness increase between treatments. Only one mutation, occurring in the internal DNA pilot protein H, was completely repeatable, and it appeared to be a necessary stepping stone toward further adaptive change. Substitutions in the surface accessible major capsid protein F appeared to be involved in capsid stability rather than specific interactions with host receptors, suggesting that non-specific alterations to capsid structure could be an important component of adaptation to novel hosts. 33% of mutations were synonymous and showed evidence of selection on codon usage. Lastly, results supported previous findings that evolving populations of small ssDNA viruses may maintain relatively high levels of genetic variation.

Efalizumab is a humanized monoclonal CD11a antibody approved for treatment of psoriasis. Its immunomodulatory effects led us study how immune responses are modified and the possible consequences for vaccinations in clinical practice. This was a randomized, single-blind, placebo-controlled, parallel-group study of 12 weeks of subcutaneous efalizumab treatment of patients with moderate psoriasis. Bacteriophage phiX174 was used as a model neoantigen to assess T-cell-dependent humoral immunity. Tetanus booster vaccine, pneumococcal vaccine, and intracutaneous skin tests were administered to further evaluate humoral and cellular immune responses. During efalizumab treatment, both primary and secondary antibody responses to phiX174, including IgM/IgG isotype switch, were reduced. There appeared to be naïve T-cell anergy to a neoantigen (phiX174) during active CD11a blockade, without tolerance to the antigen after efalizumab withdrawal. Secondary humoral immune responses to tetanus booster during treatment were reduced, but antibody titer increases led to protective levels. Responses to pneumococcal vaccination 6 weeks after withdrawal from efalizumab were not affected. Cellular immune responses to intracutaneous recall antigens were reduced during treatment and returned to pretreatment conditions after withdrawal. These results expand our knowledge of how immune responses are modulated in humans by CD11a blockade and have implications for vaccinations of patients treated with this agent.

Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

For the microviruses and the leviviruses, bacteriophages with small single-stranded genomes, host lysis is accomplished by expression of a single gene that encodes an inhibitor of cell wall synthesis. In contrast, phages with double-stranded DNA genomes use a more complex system involving, at minimum, an endolysin, which degrades peptidoglycan, and a holin, which permeabilizes the membrane in a temporally programmed manner. To explore the basis of this difference, a chimera was created in which lysis gene E of the microvirus phiX174 replaced the entire lysis cassette of phage lambda, which includes the holin gene S and the endolysin gene R. The chimeric phage was viable but more variability was observed both in the distribution of plaque sizes and in the burst sizes of single cells, compared to the isogenic S(+) parent. Using different alleles of E, it was found the average burst size increased with the duration of the latent period, just as observed with S alleles with different lysis times. Moreover, within a set of missense E alleles, it was found that variability in lysis timing was limited and almost exclusively derived from changes in the level of E accumulation. By contrast, missense mutations in S resulted in a wide variation in lysis times that was not correlated with levels of accumulation. We suggest that the properties of greater phenotypic plasticity and lesser phenotypic variation make the function of holin proteins more genetically malleable, facilitating rapid adaptation towards a lysis time that would be optimal for changed host and environmental conditions. The inferior malleability of single-gene systems like E would restrict their occurrence to phages in which coding capacity is the overriding evolutionary constraint.