RESUMO
Bacteriophage ÏX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ÏX174 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 ÏX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ÏX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ÏX174 replication; moreover, their absence has no discernible effect on ÏX174 fecundity. These results suggest IbpA/B upregulation is a response to ÏX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Replicação Viral , Escherichia coli/virologia , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/fisiologia , Bacteriófago phi X 174/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque TérmicoRESUMO
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.
Assuntos
Antibacterianos , Biofilmes , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Bacteriófagos/genética , Bacteriófagos/fisiologia , Escherichia coli/virologia , Escherichia coli/efeitos dos fármacos , Bacteriófago phi X 174/efeitos dos fármacos , Bacteriófago phi X 174/genética , Bactérias/efeitos dos fármacos , Bactérias/virologia , MutaçãoRESUMO
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.
Assuntos
Bacteriófago phi X 174 , Proteínas do Capsídeo , DNA de Cadeia Simples , DNA Viral , Proteínas de Ligação a DNA , Evolução Molecular , Empacotamento do Genoma Viral , Bacteriófago phi X 174/química , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/crescimento & desenvolvimento , Bacteriófago phi X 174/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Sequência Conservada , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Aptidão Genética , Mutação , Fenótipo , Moldes Genéticos , Vírion/química , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/metabolismoRESUMO
Water reuse is a growing global reality. In regulating water reuse, viruses have come to the fore as key pathogens due to high shedding rates, low infectious doses, and resilience to traditional wastewater treatments. To demonstrate the high log reductions required by emerging water reuse regulations, cost and practicality necessitate surrogates for viruses for use as challenge organisms in unit process evaluation and monitoring. Bacteriophage surrogates that are mitigated to the same or lesser extent than viruses of concern are routinely used for individual unit process testing. However, the behavior of these surrogates over a multi-barrier treatment train typical of water reuse has not been well-established. Toward this aim, we performed a meta-analysis of log reductions of common bacteriophage surrogates for five treatment processes typical of water reuse treatment trains: advanced oxidation processes, chlorination, membrane filtration, ozonation, and ultraviolet (UV) disinfection. Robust linear regression was applied to identify a range of doses consistent with a given log reduction of bacteriophages and viruses of concern for each treatment process. The results were used to determine relative conservatism of surrogates. We found that no one bacteriophage was a representative or conservative surrogate for viruses of concern across all multi-barrier treatments (encompassing multiple mechanisms of virus mitigation). Rather, a suite of bacteriophage surrogates provides both a representative range of inactivation and information about the effectiveness of individual processes within a treatment train. Based on the abundance of available data and diversity of virus treatability using these five key water reuse treatment processes, bacteriophages MS2, phiX174, and Qbeta were recommended as a core suite of surrogates for virus challenge testing.
Assuntos
Bacteriófagos , Purificação da Água , Água , Bacteriófago phi X 174 , Purificação da Água/métodos , Desinfecção/métodos , LevivirusRESUMO
Studies into the viral fraction of complex microbial communities, like in the mammalian gut, have recently garnered much interest. Yet there is still no standardized protocol for extracting viruses from such samples, and the protocols that exist employ procedures that skew the viral community of the sample one way or another. The first step of the extraction pipeline often consists of the basic filtering of macromolecules and bacteria, yet even this affects the viruses in a strain-specific manner. In this study, we investigate a protocol for viral extraction based on ultrafiltration and how the choice of ultrafilter might influence the extracted viral community. Clinical samples (feces, vaginal swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4, c2, Φ6, Φ29, Φx174, and Φ2972), filtered, and quantified using spot and plaque assays to estimate the loss in recovery. The enveloped Φ6 phage is especially severely affected by the choice of filter, but also tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that the pore size of ultrafilters may affect the recovery of phages in a strain- and sample-dependent manner, suggesting the need for greater thought when selecting filters for virus extraction.
Assuntos
Bacteriófagos , Caudovirales , Microbiota , Vírus , Animais , Bacteriófago phi X 174 , MamíferosRESUMO
Phage protein E shuts down cell wall biosynthesis to kill bacteria.
Assuntos
Bactérias , Bacteriólise , Bacteriófago phi X 174 , Biossíntese de Proteínas , Proteínas Virais , Bactérias/metabolismo , Bactérias/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Parede Celular/metabolismoRESUMO
The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo-microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure-revealing features of this essential enzyme-and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.
Assuntos
Antibacterianos , Bacteriólise , Bacteriófago phi X 174 , Proteínas de Escherichia coli , Escherichia coli , Proteínas Virais , Antibacterianos/metabolismo , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Imagem Individual de Molécula , Microscopia CrioeletrônicaRESUMO
Ubiquitous and abundant in ecosystems and microbiomes, gokushoviruses constitute a Microviridae subfamily, distantly related to bacteriophages ΦX174, α3, and G4. A high-resolution cryo-EM structure of gokushovirus ΦEC6098 was determined, and the atomic model was built de novo. Although gokushoviruses lack external scaffolding and spike proteins, which extensively interact with the ΦX174 capsid protein, the core of the ΦEC6098 coat protein (VP1) displayed a similar structure. There are, however, key differences. At each ΦEC6098 icosahedral 3-fold axis, a long insertion loop formed mushroom-like protrusions, which have been noted in lower-resolution gokushovirus structures. Hydrophobic interfaces at the bottom of these protrusions may confer stability to the capsid shell. In ΦX174, the N-terminus of the capsid protein resides directly atop the 3-fold axes of symmetry; however, the ΦEC6098 N-terminus stretched across the inner surface of the capsid shell, reaching nearly to the 5-fold axis of the neighboring pentamer. Thus, this extended N-terminus interconnected pentamers on the inside of the capsid shell, presumably promoting capsid assembly, a function performed by the ΦX174 external scaffolding protein. There were also key differences between the ΦX174-like DNA-binding J proteins and its ΦEC6098 homologue VP8. As seen with the J proteins, C-terminal VP8 residues were bound into a pocket within the major capsid protein; however, its N-terminal residues were disordered, likely due to flexibility. We show that the combined location and interaction of VP8's C-terminus and a portion of VP1's N-terminus are reminiscent of those seen with the ΦX174 and α3 J proteins. IMPORTANCE There is a dramatic structural and morphogenetic divide within the Microviridae. The well-studied ΦX174-like viruses have prominent spikes at their icosahedral vertices, which are absent in gokushoviruses. Instead, gokushovirus major coat proteins form extensive mushroom-like protrusions at the 3-fold axes of symmetry. In addition, gokushoviruses lack an external scaffolding protein, the more critical of the two ΦX174 assembly proteins, but retain an internal scaffolding protein. The ΦEC6098 virion suggests that key external scaffolding functions are likely performed by coat protein domains unique to gokushoviruses. Thus, within one family, different assembly paths have been taken, demonstrating how a two-scaffolding protein system can evolve into a one-scaffolding protein system, or vice versa.
Assuntos
Capsídeo , Microviridae , Capsídeo/química , Microvirus , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Ecossistema , Microviridae/química , Microviridae/metabolismo , Bacteriófago phi X 174 , Montagem de VírusRESUMO
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. ÏX174-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 ÏXG4J chimera, in which the ÏX174 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, ÏXα3J. Unlike ÏXG4J, ÏXα3J 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 ÏX174 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.
Assuntos
Bacteriófago phi X 174/genética , Proteínas do Capsídeo/genética , Capsídeo/metabolismo , DNA de Cadeia Simples/genética , DNA Viral/genética , Genoma Viral , Montagem de Vírus , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Empacotamento do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/virologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , VírionRESUMO
Bacteriophage plaque size measurement is essential for phage characterisation, but 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 and turbid 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.
Assuntos
Bacteriófago phi X 174/crescimento & desenvolvimento , Bacteriófagos/crescimento & desenvolvimento , Ensaio de Placa Viral/métodos , Automação Laboratorial , Bacteriófago phi X 174/ultraestrutura , Bacteriófagos/ultraestrutura , Processamento de Imagem Assistida por Computador , Reprodutibilidade dos Testes , SoftwareRESUMO
Recent advances in DNA sequencing open prospects to make whole-genome analysis rapid and reliable, which is promising for various applications including personalized medicine. However, existing techniques for de novo genome assembly, which is used for the analysis of genomic rearrangements, chromosome phasing, and reconstructing genomes without a reference, require solving tasks of high computational complexity. Here we demonstrate a method for solving genome assembly tasks with the use of quantum and quantum-inspired optimization techniques. Within this method, we present experimental results on genome assembly using quantum annealers both for simulated data and the [Formula: see text]X 174 bacteriophage. Our results pave a way for a significant increase in the efficiency of solving bioinformatics problems with the use of quantum computing technologies and, in particular, quantum annealing might be an effective method. We expect that the new generation of quantum annealing devices would outperform existing techniques for de novo genome assembly. To the best of our knowledge, this is the first experimental study of de novo genome assembly problems both for real and synthetic data on quantum annealing devices and quantum-inspired techniques.
Assuntos
Biologia Computacional/métodos , Genômica/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Bacteriófago phi X 174/genética , Simulação por Computador , DNA Viral/genética , Conjuntos de Dados como Assunto , Genoma Viral , Humanos , Matemática , Teoria QuânticaRESUMO
As part of a viral mitigating strategy for continuous bioprocessing, that utilizes a plug flow reactor (PFR) for continuous viral inactivation (CVI), understanding the minimum residence time as a function of reactor scale and operational conditions is critical. An empirical-based model was utilized to calculate the minimum duration a virus particle experiences within a plug flow reactor as a function of reactor design and operational conditions. This empirical model's calculations were challenged by pulse injecting the bacteriophage ΦX-174 in non-inactivating conditions and monitoring the discharge of the PFR with infectivity assays. The initial proposed empirical model, with the constraint of requiring an operational Dean number of >100, proved to be effective at calculating first breakthrough of ΦX-174 but only for the appropriate Dean number conditions. With the knowledge gained from the first empirical model, a second was generated to eliminate the Dean number constraint. This second modified empirical model proved to be successful at calculating the first breakthrough at all Dean number's tested, however CVI operation at the lower Dean's number will lead to an increased asymmetry (i.e., increased tailing) in the residence time distribution.
Assuntos
Bacteriófago phi X 174 , Reatores Biológicos , Modelos Biológicos , Inativação de VírusRESUMO
Bactofection, a bacterial-mediated form of genetic transfer, is highlighted as an alternative mechanism for gene therapy. A key advantage of this system for immune-reactivity purposes stems from the nature of the bacterial host capable of initiating an immune response by attracting recognition and cellular uptake by antigen-presenting cells (APCs). The approach is also a suitable technique to deliver larger genetic constructs more efficiently as it can transfer plasmids of varying sizes into target mammalian cells. Given these advantages, bacterial vectors are being studied as potential carriers for the delivery of plasmid DNA into target cells to enable expression of heterologous proteins. The bacteria used for bactofection are generally nonpathogenic; however, concerns arise due to the use of a biological agent. To overcome such concerns, enhanced bacterial degradation has been engineered as an attenuation and safety feature for bactofection vectors. In particular, the ΦX174 lysis E (LyE) gene can be repurposed to both minimize bacterial survival within mammalian hosts while also improving overall gene delivery. More specifically, an engineered bacterial vector carrying the LyE gene showed improved gene delivery and safety profiles when tested with murine RAW264.7 macrophage APCs. This chapter outlines steps taken to engineer E. coli for LyE expression as a safer and more effective genetic antigen delivery bactofection vehicle in the context of vaccine utility.
Assuntos
Bacteriófago phi X 174/fisiologia , Escherichia coli/virologia , Expressão Gênica , Transdução Genética , Proteínas Virais/genética , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hemólise , Interações Hospedeiro-Patógeno , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , Plasmídeos/genéticaRESUMO
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.
Assuntos
Antibiose/genética , Bacteriófago phi X 174/genética , Proteínas de Escherichia coli/genética , Escherichia coli/virologia , Lisogenia/genética , Peptidilprolil Isomerase/genética , Toxinas Biológicas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago phi X 174/metabolismo , Bacteriófago phi X 174/patogenicidade , Sítios de Ligação , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/virologia , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Nanopartículas/química , Peptidilprolil Isomerase/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solubilidade , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
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.
Assuntos
Linfócitos B/citologia , Bacteriófago phi X 174/imunologia , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Adolescente , Adulto , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Ligante de CD40/deficiência , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Proteínas I-kappa B/genética , Imunização , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Síndromes de Imunodeficiência/patologia , Memória Imunológica/genética , Memória Imunológica/imunologia , Lactente , Masculino , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
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.
Assuntos
Bacteriófago phi X 174/genética , Códon , Genoma Viral , Epistasia Genética , Genes Virais , Aptidão Genética , Modelos Genéticos , Seleção Genética , Vacinas ViraisRESUMO
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.
Assuntos
Bacteriófago phi X 174/metabolismo , Levivirus/metabolismo , Viabilidade Microbiana , SARS-CoV-2/metabolismo , Saliva/virologia , Bacteriófago phi 6/metabolismo , COVID-19/transmissão , Microbiologia Ambiental , Humanos , Ensaio de Placa Viral , Inativação de VírusRESUMO
Bacteriophage ÏX174 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 ÏX174 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 ÏX174 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.
Assuntos
Bacteriófago phi X 174/genética , Transcrição Gênica , Bacteriófago phi X 174/metabolismo , Sequência de Bases , Escherichia coli/virologia , Regulação Viral da Expressão Gênica , Regiões Promotoras Genéticas , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Bacteriophage ÏX174 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 Å-long section near the tube's C-terminus that does not exhibit this motif. The majority of the inward-facing residues in this region are conserved across the three ÏX174-like clades, suggesting that they play an important role during genome delivery. To test this hypothesis, and explore the general function of the tube'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'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's inward facing glutamine residues exert a frictional force, or drag, that controls genome release.
Assuntos
Bacteriófago phi X 174/genética , Proteínas do Capsídeo/genética , DNA Viral/metabolismo , Proteínas da Cauda Viral/genética , Internalização do Vírus , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Cristalografia por Raios X , DNA Viral/genética , Genoma Viral/genética , Mutagênese , Proteínas da Cauda Viral/metabolismoRESUMO
Sunlight-mediated inactivation of microorganisms is a low-cost approach to disinfect drinking water and wastewater. The reactions involved are affected by a wide range of factors, and a lack of knowledge about their relative importance makes it challenging to optimize treatment systems. To characterize the relative importance of environmental conditions, photoreactivity, water quality, and engineering design in the sunlight inactivation of viruses, we modeled the inactivation of three-human adenovirus and two bacteriophages-MS2 and phiX174-in surface waters and waste stabilization ponds by integrating solar irradiance and aquatic photochemistry models under uncertainty. Through global sensitivity analyses, we quantitatively apportioned the variability of predicted sunlight inactivation rate constants to different factors. Most variance was associated with the variability in and interactions among time, location, nonpurgeable organic carbon (NPOC) concentration, and pond depth. The photolysis quantum yield of the virus outweighed the seasonal solar motion in the impact on inactivation rates. Further, comparison of simulated sunlight inactivation efficacy in maturation ponds under different design decisions showed that reducing pond depth can increase the log inactivation at the cost of larger land area, but increasing hydraulic retention time by adding ponds in series yielded greater improvements in inactivation.