Role of Phage Capsid in the Resistance to UV-C Radiations.

The conformational variation of the viral capsid structure plays an essential role both for the environmental resistance and acid nuclear release during cellular infection. The aim of this study was to evaluate how capsid rearrangement in engineered phages of M13 protects viral DNA and peptide bonds from damage induced by UV-C radiation. From in silico 3D modelling analysis, two M13 engineered phage clones, namely P9b and 12III1, were chosen for (i) chemical features of amino acids sequences, (ii) rearrangements in the secondary structure of their pVIII proteins and (iii) in turn the interactions involved in phage capsid. Then, their resistance to UV-C radiation and hydrogen peroxide (H2O2) was compared to M13 wild-type vector (pC89) without peptide insert. Results showed that both the phage clones acquired an advantage against direct radiation damage, due to a reorganization of interactions in the capsid for an increase of H-bond and steric interactions. However, only P9b had an increase in resistance against H2O2. These results could help to understand the molecular mechanisms involved in the stability of new virus variants, also providing quick and necessary information to develop effective protocols in the virus inactivation for human activities, such as safety foods and animal-derived materials.

Self-Assembled Phage-Based Colloids for High Localized Enzymatic Activity.

Catalytically active colloids are model systems for chemical motors and active matter. It is desirable to replace the inorganic catalysts and the toxic fuels that are often used with biocompatible enzymatic reactions. However, compared to inorganic catalysts, enzyme-coated colloids tend to exhibit less activity. Here, we show that the self-assembly of genetically engineered M13 bacteriophages that bind enzymes to magnetic beads ensures high and localized enzymatic activity. These phage-decorated colloids provide a proteinaceous environment for directed enzyme immobilization. The magnetic properties of the colloidal carrier particle permit repeated enzyme recovery from a reaction solution, while the enzymatic activity is retained. Moreover, localizing the phage-based construct with a magnetic field in a microcontainer allows the enzyme-phage-colloids to function as an enzymatic micropump, where the enzymatic reaction generates a fluid flow. This system shows the fastest fluid flow reported to date by a biocompatible enzymatic micropump. In addition, it is functional in complex media including blood, where the enzyme-driven micropump can be powered at the physiological blood-urea concentrations.

Visible-light-driven photocatalytic inactivation of bacteriophage f2 by Cu-TiO2 nanofibers in the presence of humic acid.

Pathogenic viruses in drinking water are great threats to public health. Visible-light-driven photocatalysis is a promising technology for virus inactivation. However, the existing photocatalytic antiviral research studies have mostly been carried out in single-component systems, neglecting the effect of natural organic matter, which exists widely in actual water bodies. In this paper, electrospun Cu-TiO2 nanofibers were prepared as photocatalysts, and their photocatalytic antiviral performance in the presence of humic acid (HA) was comprehensively studied for the first time. The properties of the reaction mixture were measured during the reaction. In addition, the safety, reliability and stability of photocatalytic disinfection in the mixed system were evaluated. The results showed that the virus removal efficiency decreased with the increase of the HA concentration. The type of reaction solution, such as PBS buffer solution or water, did not affect the removal efficiency noticeably. Under acidic conditions, the electrostatic forces between photocatalysts and viruses were strengthened, leading to higher virus removal efficiency. As the reaction time went on, the pH value in the solution increased first and then tended to be stable, the conductivity remained stable, and the dissolved oxygen increased first and then decreased. The safety test showed that the concentration of Cu ions released into the solution was lower than specified by the international standards. No photoreactivation was observed, and the addition of HA significantly reduced the reutilization efficiency of the photocatalysts.

Cytotoxic and mutagenic properties of O6-alkyl-2'-deoxyguanosine lesions in Escherichia coli cells.

Environmental exposure and cellular metabolism can give rise to DNA alkylation, which can occur on the nitrogen and oxygen atoms of nucleobases, as well as on the phosphate backbone. Although O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are known to be associated with cancer, not much is known about how the alkyl group structures in these lesions affect their repair and replicative bypass in vivo or how translesion synthesis DNA polymerases influence the latter process. To answer these questions, here we synthesized oligodeoxyribonucleotides harboring seven O6-alkyl-dG lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, or sBu, and examined the impact of these lesions on DNA replication in Escherichia coli cells. We found that replication past all the O6-alkyl-dG lesions was highly efficient and that SOS-induced DNA polymerases play redundant roles in bypassing these lesions. Moreover, these lesions directed exclusively the G → A mutation, the frequency of which increased with the size of the alkyl group on the DNA. This could be attributed to the varied repair efficiencies of these lesions by O6-alkylguanine DNA alkyltransferase (MGMT) in cells, which involve the MGMT Ogt and, to a lesser extent, Ada. In conclusion, our study provides important new knowledge about the repair of the O6-alkyl-dG lesions and their recognition by the E. coli DNA replication machinery. Our results suggest that the lesions' carcinogenic potentials may be attributed, at least in part, to their strong mutagenic potential and their efficient bypass by the DNA replication machinery.

Dual UV irradiation-based metal oxide nanoparticles for enhanced antimicrobial activity in Escherichia coli and M13 bacteriophage.

Metal oxide (MO) nanoparticles have been studied as nano-antibiotics due to their antimicrobial activities even in antibiotic-resistant microorganisms. We hypothesized that a hybrid system of dual UV irradiation and MO nanoparticles would have enhanced antimicrobial activities compared with UV or MO nanoparticles alone. In this study, nanoparticles of ZnO, ZnTiO3, MgO, and CuO were selected as model nanoparticles. A dual UV collimated beam device of UV-A and UV-C was developed depending upon the lamp divided by coating. Physicochemical properties of MO nanoparticles were determined using powder X-ray diffractometry (PXRD), Brunauer-Emmett-Teller analysis, and field emission-scanning electron microscopy with energy-dispersive X-ray spectroscopy. Atomic force microscopy with an electrostatic force microscopy mode was used to confirm the surface topology and electrostatic characteristics after dual UV irradiation. For antimicrobial activity test, MO nanoparticles under dual UV irradiation were applied to Escherichia coli and M13 bacteriophage (phage). The UV-A and UV-C showed differential intensities in the coated and uncoated areas (UV-A, coated = uncoated; UV-C, coated ≪ uncoated). MO nanoparticles showed sharp peaks in PXRD patterns, matched to pure materials. Their primary particle sizes were less than 100 nm with irregular shapes, which had an 8.6~25.6 m2/g of specific surface area with mesopores of 22~262 nm. The electrostatic properties of MO nanoparticles were modulated after UV irradiation. ZnO, MgO, and CuO nanoparticles, except ZnTiO3 nanoparticles, showed antibacterial effects on E. coli. Antimicrobial effects on E. coli and phages were also enhanced after cyclic exposure of dual UV and MO nanoparticle treatment using the uncoated area, except ZnO nanoparticles. Our results demonstrate that dual UV-MO nanoparticle hybrid system has a potential for disinfection. We anticipate that it can be developed as a next-generation disinfection system in pharmaceutical industries and water purification systems.

Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation.

N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA.

Production and Purification of Recombinant Filamentous Bacteriophages Displaying Immunogenic Heterologous Epitopes.

Viruslike particles often combine high physical stability with robust immunogenicity. Furthermore, when such particles are based on bacteriophages, they can be produced in high amounts at minimal cost and typically will require only standard biologically contained facilities. We provide protocols for the characterization and purification of recombinant viruslike particles derived from filamentous bacteriophages. As an example, we focus on filamentous Escherichia coli fd phage displaying a conserved influenza A virus epitope that is fused genetically to the N-terminus of the major coat protein of this phage. A step-by-step procedure to obtain a high-titer, pure recombinant phage preparation is provided. We also describe a quality control experiment based on a biological readout of the purified fd phage preparation. These protocols together with the highlighted critical steps may facilitate generic implementation of the provided procedures for the display of other epitopes by recombinant fd phages.

Tautomerism provides a molecular explanation for the mutagenic properties of the anti-HIV nucleoside 5-aza-5,6-dihydro-2'-deoxycytidine.

Viral lethal mutagenesis is a strategy whereby the innate immune system or mutagenic pool nucleotides increase the error rate of viral replication above the error catastrophe limit. Lethal mutagenesis has been proposed as a mechanism for several antiviral compounds, including the drug candidate 5-aza-5,6-dihydro-2'-deoxycytidine (KP1212), which causes A-to-G and G-to-A mutations in the HIV genome, both in tissue culture and in HIV positive patients undergoing KP1212 monotherapy. This work explored the molecular mechanism(s) underlying the mutagenicity of KP1212, and specifically whether tautomerism, a previously proposed hypothesis, could explain the biological consequences of this nucleoside analog. Establishing tautomerism of nucleic acid bases under physiological conditions has been challenging because of the lack of sensitive methods. This study investigated tautomerism using an array of spectroscopic, theoretical, and chemical biology approaches. Variable temperature NMR and 2D infrared spectroscopic methods demonstrated that KP1212 existed as a broad ensemble of interconverting tautomers, among which enolic forms dominated. The mutagenic properties of KP1212 were determined empirically by in vitro and in vivo replication of a single-stranded vector containing a single KP1212. It was found that KP1212 paired with both A (10%) and G (90%), which is in accord with clinical observations. Moreover, this mutation frequency is sufficient for pushing a viral population over its error catastrophe limit, as observed before in cell culture studies. Finally, a model is proposed that correlates the mutagenicity of KP1212 with its tautomeric distribution in solution.

Rheology and DWS microrheology of concentrated suspensions of the semiflexible filamentous fd virus.

Microrheology measurements were performed on suspensions of bacteriophage fd with diffusive wave spectroscopy in the concentrated regime, at different values of ionic strength. Viscosity vs. shear rate was also measured, and the effect of bacteriophage concentration and salt addition on shear thinning was determined, as well as on the peaks in the viscosity vs. shear curves corresponding to a transition from tumbling to wagging flow. The influence of concentration and salt addition on the mean square displacement of microspheres embedded in the suspensions was determined, as well as on their viscoelastic moduli up to high angular frequencies. Our results were compared with another microrheology technique previously reported where the power spectral density of thermal fluctuations of embedded micron-sized particles was evaluated. Although both results in general agree, the diffusive wave spectroscopy results are much less noisy and can reach larger frequencies. A comparison was made between measured and calculated shear modulus. Calculations were made employing the theory for highly entangled isotropic solutions of semiflexible polymers using a tube model, where various ways of calculating the needed parameters were used. Although some features are captured by the model, it is far from the experimental results mainly at high frequencies.

Supersaturated dispersions of rodlike viruses with added attraction.

The kinetics of isotropic-nematic (I-N) and nematic-isotropic (N-I) phase transitions in dispersions of rodlike fd viruses are studied. Concentration quenches were applied using pressure jumps in combination with polarization microscopy, birefringence, and turbidity measurements. The full biphasic region could be accessed, resulting in the construction of an experimental analog of the bifurcation diagram. The N-I spinodal points for dispersions of rods with varying concentrations of depletion agent (dextran) were obtained from orientation quenches using cessation of shear flow in combination with small-angle light scattering. We found that the location of the N-I spinodal point is independent of the attraction, which was confirmed by theory. Surprisingly, the experiments showed that also the absolute induction time, the critical nucleus, and the growth rate are insensitive of the attraction if the concentration is scaled to the distance to the phase boundaries.

Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pI target proteins.

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Intracellular biogenic silver nanoparticles for the generation of carbon supported antiviral and sustained bactericidal agents.

Intracellular silver nanoparticles produced by exposing silver ions to the fungus Aspergillus ochraceus were heat-treated in nitrogen environment to yield silver nanoparticles embedded in carbonaceous supports. This carbonaceous matrix embedded silver nanoparticles showed antimicrobial properties against both bacteria (Gram-positive and Gram-negative) and virus (M 13 phage virus). The bactericidal effects were noticed even after washing and repeated exposure of these carbon supported silver nanoparticles to fresh bacterial cultures, revealing their sustained activity.

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA.

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.

Inhibition of bacteriophage m13 replication with esterified milk proteins.

Esterified milk proteins [methylated (Met) or ethylated (Et) alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), and beta-casein (BCN)], unmodified native milk proteins, and native basic proteins (calf thymus histone and hen egg white lysozyme) were tested for their antiviral activity against the bacteriophage M13 and for their influence on its replication (except BCN). All esterified milk proteins showed an antiviral activity against the bacteriophage M13, proportional to the extent of esterification and, hence, to the increased basicity of the modified proteins. Antiviral activity of 100% Met-BLG disappeared after its pepsinolysis but not after its trypsinolysis. The antiviral activity of Met-BLG was much higher than that of native basic proteins (histone and lysozyme). One hundred percent Met-BLG and 73% Et-BLG inhibited the replication of bacteriophage M13 completely, whereas 60% Met-ALA inhibited phage replication partially. Calf thymus histone inhibited the replication of bacteriophage M13 at a lower extent (20%) than Met- and Et-BLG (100% inhibition). Protein concentration, pH, and concentration of the Escherichia coli culture in the preincubation medium of the virus were other factors influencing antiviral activity. Interactions of esterified proteins with the phage DNA (phenol extracted) followed the same pattern as observed during studies of the inhibition of the phage replication: Met-BLG > Et-BLG > or = Met-ALA.

Cobalt ion mediated self-assembly of genetically engineered bacteriophage for biomimetic Co-Pt hybrid material.

Biological scaffolds are used for the synthesis of inorganic materials due to their ability to self-assemble and nucleate crystal formation. We report the self-assembly of engineered M13 bacteriophage as a template for Co-Pt crystals. A M13 phage library with an octapeptide library on the major coat protein (pVIII) was used for selection of binders to cobalt ions. Fibrous structures with directionally ordered M13 phage were obtained by interaction with cobalt ions. Co-Pt alloys were synthesized on the fibrous scaffold, and their magnetic properties were characterized. The mineralization showed organized nanoparticles on fibrous bundles. This approach using the phage pVIII library allows for genetic selection that both induces assembly of the phage and directs mineralization of the selected inorganic material.

Two-triplet CGA repeats impede DNA replication in bacteriophage M13 in Escherichia coli.

Expansion and contraction instabilities associated with CAG, CGG, GAA and CGA (GAC) repeats propagation cause more than a dozen human genetic diseases and cancers. In this work, the propagation behavior of a bacteriophage M13 carrying a calf prochymosin cDNA fragment with a (CGA)2 repeat in a small hairpin forming region is reported. Such a M13 derivative when propagated in Escherichia coli, produces small plaques by decreasing phage yield and also mitigates the inhibition on host cell growth, compared to those control bacteriophages either containing a "CTGCTA" sequence or wildtype, suggesting that CGA2 repeat impedes DNA replication in vivo. Moreover, an increased internal free energy is found associated with (CGA)2 sequence compared to those "CTGCTA" and wildtype, which ruled out a possibility of CGA2 repeat effects on propagation is through influencing the hairpin structure formation.

The effect of an agglutogen on virus infection: biotinylated filamentous phages and avidin as a model.

To address the effect of an agglutogen on virus infection, we studied the avidin-associated inhibition of infection by biotinylated M13 phages (BIO-phages). Microscopic observation of mixtures of BIO-phages and avidin-fluorescein conjugates revealed many aggregates. Even at low phage concentrations, avidin induced inhibition of infection significantly. Anti-M13 phage antibody also made aggregates and inhibited the infection but in a different manner from avidin. The inhibition by avidin was at > or = 2 microg/ml, time dependent and marked until 10 min after the mixing of the BIO-phages and Escherichia coli. On the other hand, antibody inhibited the infection at > or = 0.1 microg/ml dose dependently, and the inhibition was time dependent and marked until 45 min after the mixing at moderate and low phage concentrations. These results indicate that avidin against BIO-phages and antibodies are agglutogens, and the inhibition of the BIO-phages by avidin is closely related to the tetramerization of avidin. Agglutogens may be novel alternative antiviral drugs.

Novel peptides that inhibit the propagation of Newcastle disease virus.

A disulfide constrained random heptapeptide library displayed on filamentous bacteriophage M13 was applied to select specific ligands that interact with Newcastle disease virus (NDV). A fusion phage carrying the amino acid sequence TLTTKLY was selected from the panning procedure. An antibody competition assay showed that the selected phage was capable of competing with the polyclonal antibodies raised against NDV for binding sites on the virus. Determination of the binding affinity of this phage with NDV by an equilibrium binding assay in solution revealed two different dissociation constants, suggesting that there could be two distinct binding sites for the phage on NDV. Synthetic peptides with the sequence CTLTTKLYC, either in linear or cyclic conformations inhibited the binding of phage bearing the same sequence to NDV. These peptides also inhibited the hemolytic activity of the virus as well as its propagation in embryonated chicken eggs.

Oxidative damage and induced mutations in m13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation.

N:-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes alpha-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations. Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2. In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA. Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed. Singlet oxygen is likely to account for the formation of 8-oxodGuo. We analyzed the spectrum of mutations in lacZalpha of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA. The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts. A higher level of mutation was observed with the mutM-deficient host than with the -proficient host. Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts. With the mutM-deficient host, we observed an overall increase in the percentage of GC-->TA transversions. In addition we noted that there were fewer GC-->AT transitions than in the mutM-proficient host. With these hosts, different hot spots were observed and a new GC-->TA hot spot was produced. The formation of 8-oxodGuo in DNA, which is known to induce GC-->TA transversion, may contribute to mutagenesis by NPYR plus UVA.

The efficiency of Escherichia coli selenocysteine insertion is influenced by the immediate downstream nucleotide.

Selenocysteine (Sec) incorporation requires the TGA opal codon and a downstream Sec insertion sequence (SECIS), which can be partially randomized and cloned into M13 pIII fusion constructs for phage display. This combinatorial approach provides a convenient non-radioactive assay that couples phage production to opal suppression. Two SECIS libraries were prepared, with the immediate downstream nucleotide either randomized (TGAN) or fixed as thymidine (TGAT). The TGAN library resulted in a majority of clones with a downstream purine and selenium-independent phage production, implicating the endo-genous tryptophan-inserting opal suppression pathway. Although the addition of sodium selenite to the growth medium did not affect phage production, it did increase the level of Sec insertion, as shown by the chemical reactivity of the resulting phage. The TGAT phage library yielded clones with strictly selenium-dependent phage production and reactivity consistent with the presence of Sec. These clones were prone to spontaneous mutation upon further propagation, however, resulting in loss of the selenium-dependent phenotype. We conclude that the immediate downstream nucleotide determines whether the endogenous opal suppression pathway competes with co-translational Sec insertion.