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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.

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.

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.

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.

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.

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 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.

Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids.

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.

Acupuncture sample injection is a simple method to deliver well-defined nanoliter-scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost-effective acupuncture sample injection method can be used for PDMS microchip-based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA-HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.

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.

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.

DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.

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.

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

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.

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.