Disenfranchised DNA: biochemical analysis of mutant øX174 DNA-binding proteins may further elucidate the evolutionary significance of the unessential packaging protein A.

Most icosahedral DNA viruses package and condense their genomes into pre-formed, volumetrically constrained capsids. However, concurrent genome biosynthesis and packaging are specific to single-stranded (ss) DNA micro- and parvoviruses. Before packaging, ~120 copies of the øX174 DNA-binding protein J interact with double-stranded DNA. 60 J proteins enter the procapsid with the ssDNA genome, guiding it between 60 icosahedrally ordered DNA-binding pockets formed by the capsid proteins. Although J proteins are small, 28-37 residues in length, they have two domains. The basic, positively charged N-terminus guides the genome between binding pockets, whereas the C-terminus acts as an anchor to the capsid's inner surface. Three C-terminal aromatic residues, W30, Y31, and F37, interact most extensively with the coat protein. Their corresponding codons were mutated, and the resulting strains were biochemically and genetically characterized. Depending on the mutation, the substitutions produced unstable packaging complexes, unstable virions, infectious progeny, or particles packaged with smaller genomes, the latter being a novel phenomenon. The smaller genomes contained internal deletions. The juncture sequences suggest that the unessential A* (A star) protein mediates deletion formation.IMPORTANCEUnessential but strongly conserved gene products are understudied, especially when mutations do not confer discernable phenotypes or the protein's contribution to fitness is too small to reliably determine in laboratory-based assays. Consequently, their functions and evolutionary impact remain obscure. The data presented herein suggest that microvirus A* proteins, discovered over 40 years ago, may hasten the termination of non-productive packaging events. Thus, performing a salvage function by liberating the reusable components of the failed packaging complexes, such as DNA templates and replication enzymes.

Antibacterial Potential of Non-Tailed Icosahedral Phages Alone and in Combination with Antibiotics.

Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.

The Effects of Packaged, but Misguided, Single-Stranded DNA Genomes Are Transmitted to the Outer Surface of the ϕX174 Capsid.

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.

A lab-on-a-chip for free-flow electrophoretic preconcentration of viruses and gel electrophoretic DNA extraction.

Nucleic acid amplification techniques such as real-time PCR are essential instruments for the identification and quantification of viruses. They are fast, very sensitive and highly specific, but often require elaborate and labor intensive sample preparation to achieve successful amplification of the target sequence. In this work we demonstrate the complete microfluidic preparation of amplifiable virus DNA from dilute specimens. Our approach combines free-flow electrophoretic preconcentration of viral particles with thermal lysis and gel-electrophoretic nucleic acid extraction on a single device. The on-chip preconcentration achieves a capture efficiency of >99% for dilute suspensions of bacteriophage PhiX174. Following preconcentration, phages are thermally lysed and released DNA is recovered after 40 s of on-chip gel-electrophoresis with a recovery rate of ∼73%. Furthermore we demonstrate a detection limit of ∼1 PFU ml-1 (∼0.02 DNA copies per µl) for the detection of bacteriophage PhiX174 by PCR. To simplify operation of the device, we describe the development of a custom-made chip holder as well as a compact peristaltic pump and power supply, which enable user-friendly operation with low risk of cross-contamination and high potential for automation in the field of point-of-care diagnostics.

Generation and immunity effect evaluation of biotechnology-derived Aeromonas veronii ghost by PhiX174 gene E-mediated inactivation in koi (Cyprinus carprio koi).

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 ±â€¯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.

Coat Protein Mutations That Alter the Flux of Morphogenetic Intermediates through the ϕX174 Early Assembly Pathway.

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.

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

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.

Heterogeneous asymmetric recombinase polymerase amplification (haRPA) for rapid hygiene control of large-volume water samples.

Hygiene of drinking water is periodically controlled by cultivation and enumeration of indicator bacteria. Rapid and comprehensive measurements of emerging pathogens are of increasing interest to improve drinking water safety. In this study, the feasibility to detect bacteriophage PhiX174 as a potential indicator for virus contamination in large volumes of water is demonstrated. Three consecutive concentration methods (continuous ultrafiltration, monolithic adsorption filtration, and centrifugal ultrafiltration) were combined to concentrate phages stepwise from 1250 L drinking water into 1 mL. Heterogeneous asymmetric recombinase polymerase amplification (haRPA) is applied as rapid detection method. Field measurements were conducted to test the developed system for hygiene online monitoring under realistic conditions. We could show that this system allows the detection of artificial contaminations of bacteriophage PhiX174 in drinking water pipelines.

Structure-Function Analysis of the ϕX174 DNA-Piloting Protein Using Length-Altering Mutations.

UNLABELLED: 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.

Acupuncture injection for field amplified sample stacking and glass microchip-based capillary gel electrophoresis.

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.

Empirical estimation of sequencing error rates using smoothing splines.

BACKGROUND: Next-generation sequencing has been used by investigators to address a diverse range of biological problems through, for example, polymorphism and mutation discovery and microRNA profiling. However, compared to conventional sequencing, the error rates for next-generation sequencing are often higher, which impacts the downstream genomic analysis. Recently, Wang et al. (BMC Bioinformatics 13:185, 2012) proposed a shadow regression approach to estimate the error rates for next-generation sequencing data based on the assumption of a linear relationship between the number of reads sequenced and the number of reads containing errors (denoted as shadows). However, this linear read-shadow relationship may not be appropriate for all types of sequence data. Therefore, it is necessary to estimate the error rates in a more reliable way without assuming linearity. We proposed an empirical error rate estimation approach that employs cubic and robust smoothing splines to model the relationship between the number of reads sequenced and the number of shadows. RESULTS: We performed simulation studies using a frequency-based approach to generate the read and shadow counts directly, which can mimic the real sequence counts data structure. Using simulation, we investigated the performance of the proposed approach and compared it to that of shadow linear regression. The proposed approach provided more accurate error rate estimations than the shadow linear regression approach for all the scenarios tested. We also applied the proposed approach to assess the error rates for the sequence data from the MicroArray Quality Control project, a mutation screening study, the Encyclopedia of DNA Elements project, and bacteriophage PhiX DNA samples. CONCLUSIONS: The proposed empirical error rate estimation approach does not assume a linear relationship between the error-free read and shadow counts and provides more accurate estimations of error rates for next-generation, short-read sequencing data.

The Kinetic and Thermodynamic Aftermath of Horizontal Gene Transfer Governs Evolutionary Recovery.

Shared host cells can serve as melting pots for viral genomes, giving many phylogenies a web-like appearance due to horizontal gene transfer. However, not all virus families exhibit web-like phylogenies. Microviruses form three distinct clades, represented by φX174, G4, and α3. Here, we investigate protein-based barriers to horizontal gene transfer between clades. We transferred gene G, which encodes a structural protein, between φX174 and G4, and monitored the evolutionary recovery of the resulting chimeras. In both cases, particle assembly was the major barrier after gene transfer. The G4φXG chimera displayed a temperature-sensitive assembly defect that could easily be corrected through single mutations that promote productive assembly. Gene transfer in the other direction was more problematic. The initial φXG4G chimera required an exogenous supply of both the φX174 major spike G and DNA pilot H proteins. Elevated DNA pilot protein levels may be required to compensate for off-pathway reactions that may have become thermodynamically and/or kinetically favored when the foreign spike protein was present. After three targeted genetic selections, the foreign spike protein was productively integrated into the φX174 background. The first adaption involved a global decrease in gene expression. This was followed by modifications affecting key protein-protein interactions that govern assembly. Finally, gene expression was re-elevated. Although the first selection suppresses nonproductive reactions, subsequent selections promote productive assembly and ultimately viability. However, viable chimeric strains exhibited reduced fitness compared with wild-type. This chimera's path to recovery may partially explain how unusual recombinant viruses could persist long enough to naturally emerge.

High-resolution structure of a virally encoded DNA-translocating conduit and the mechanism of DNA penetration.

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-Šresolution H-tube crystal structure suggests functional and energetic mechanisms that may be common features of DNA transport through virally encoded conduits.

Mutations in the N terminus of the oX174 DNA pilot protein H confer defects in both assembly and host cell attachment.

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.

An empirical test of the mutational landscape model of adaptation using a single-stranded DNA virus.

The primary impediment to formulating a general theory for adaptive evolution has been the unknown distribution of fitness effects for new beneficial mutations. By applying extreme value theory, Gillespie circumvented this issue in his mutational landscape model for the adaptation of DNA sequences, and Orr recently extended Gillespie's model, generating testable predictions regarding the course of adaptive evolution. Here we provide the first empirical examination of this model, using a single-stranded DNA bacteriophage related to phiX174, and find that our data are consistent with Orr's predictions, provided that the model is adjusted to incorporate mutation bias. Orr's work suggests that there may be generalities in adaptive molecular evolution that transcend the biological details of a system, but we show that for the model to be useful as a predictive or inferential tool, some adjustments for the biology of the system will be necessary.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

Adaptive regulatory substitutions affect multiple stages in the life cycle of the bacteriophage φX174.

BACKGROUND: Previously, we showed that adaptive substitutions in one of the three promoters of the bacteriophage φX174 improved fitness at high-temperature by decreasing transcript levels three- to four-fold. To understand how such an extreme change in gene expression might lead to an almost two-fold increase in fitness at the adaptive temperature, we focused on stages in the life cycle of the phage that occur before and after the initiation of transcription. For both the ancestral strain and two single-substitution strains with down-regulated transcription, we measured seven phenotypic components of fitness (attachment, ejection, eclipse, virion assembly, latent period, lysis rate and burst size) during a single cycle of infection at each of two temperatures. The lower temperature, 37°C, is the optimal temperature at which phages are cultivated in the lab; the higher temperature, 42°C, exerts strong selection and is the condition under which these substitutions arose in evolution experiments. We augmented this study by developing an individual-based stochastic model of this same life cycle to explore potential explanations for our empirical results. RESULTS: Of the seven fitness parameters, three showed significant differences between strains that carried an adaptive substitution and the ancestor, indicating the presence of pleiotropy in regulatory evolution. 1) Eclipse was longer in the adaptive strains at both the optimal and high-temperature environments. 2) Lysis rate was greater in the adaptive strains at the high temperature. 3) Burst size for the mutants was double that of the ancestor at the high temperature, but half that at the lower temperature. Simulation results suggest that eclipse length and latent period variance can explain differences in burst sizes and fitness between the mutant and ancestral strains. CONCLUSIONS: Down-regulating transcription affects several steps in the phage life cycle, and all of these occur after the initiation of transcription. We attribute the apparent tradeoff between delayed progeny production and faster progeny release to improved host resource utilization at high temperature.

Detecting heteroplasmy from high-throughput sequencing of complete human mitochondrial DNA genomes.

Heteroplasmy, the existence of multiple mtDNA types within an individual, has been previously detected by using mostly indirect methods and focusing largely on just the hypervariable segments of the control region. Next-generation sequencing technologies should enable studies of heteroplasmy across the entire mtDNA genome at much higher resolution, because many independent reads are generated for each position. However, the higher error rate associated with these technologies must be taken into consideration to avoid false detection of heteroplasmy. We used simulations and phiX174 sequence data to design criteria for accurate detection of heteroplasmy with the Illumina Genome Analyzer platform, and we used artificial mixtures and replicate data to test and refine the criteria. We then applied these criteria to mtDNA sequence reads for 131 individuals from five Eurasian populations that had been generated via a parallel tagged approach. We identified 37 heteroplasmies at 10% frequency or higher at 34 sites in 32 individuals. The mutational spectrum does not differ between heteroplasmic mutations and polymorphisms in the same individuals, but the relative mutation rate at heteroplasmic mutations is significantly higher than that estimated for all mutable sites in the human mtDNA genome. Moreover, there is also a significant excess of nonsynonymous mutations observed among heteroplasmies, compared to polymorphism data from the same individuals. Both mutation-drift and negative selection influence the fate of heteroplasmies to determine the polymorphism spectrum in humans. With appropriate criteria for avoiding false positives due to sequencing errors, next-generation technologies can provide novel insights into genome-wide aspects of mtDNA heteroplasmy.

Conformational switch-defective X174 internal scaffolding proteins kinetically trap assembly intermediates before procapsid formation.

Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, X174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.

Nucleoside analogue mutagenesis of a single-stranded DNA virus: evolution and resistance.

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 X174 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 X174 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.