The inhibition of infectivity of bacteriophage phi X174 by high-valency metal cations and cyclic polyamines.
Ontology highlight
ABSTRACT:
SUBMITTER: Rowatt E
PROVIDER: S-EPMC1148180 | biostudies-other | 1987 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Like many viruses, bacteriophage phi X174 packages its DNA genome into a procapsid that is assembled from structural intermediates and scaffolding proteins. The procapsid contains the structural proteins F, G and H, as well as the scaffolding proteins B and D. Provirions are formed by packaging of DNA together with the small internal J proteins, while losing at least some of the B scaffolding proteins. Eventually, loss of the D scaffolding proteins and the remaining B proteins leads to the formation of mature virions.phi X174 108S 'procapsids' have been purified in milligram quantities by removing 114S (mature virion) and 70S (abortive capsid) particles from crude lysates by differential precipitation with polyethylene glycol. 132S 'provirions' were purified on sucrose gradients in the presence of EDTA. Cryo-electron microscopy (cryo-EM) was used to obtain reconstructions of procapsids and provirions. Although these are very similar to each other, their structures differ greatly from that of the virion. The F and G proteins, whose atomic structures in virions were previously determined from X-ray crystallography, were fitted into the cryo-EM reconstructions. This showed that the pentamer of G proteins on each five-fold vertex changes its conformation only slightly during DNA packaging and maturation, whereas major tertiary and quaternary structural changes occur in the F protein. The procapsids and provirions were found to contain 120 copies of the D protein arranged as tetramers on the two-fold axes. DNA might enter procapsids through one of the 30 A diameter holes on the icosahedral three-fold axes.Combining cryo-EM image reconstruction and X-ray crystallography has revealed the major conformational changes that can occur in viral assembly. The function of the scaffolding proteins may be, in part, to support weak interactions between the structural proteins in the procapsids and to cover surfaces that are subsequently required for subunit-subunit interaction in the virion. The structures presented here are, therefore, analogous to chaperone proteins complexed with folding intermediates of a substrate.
| S-EPMC4142697 | biostudies-other
Project description:Gene regulation plays a central role in the adaptation of organisms to their environments. There are many molecular components to gene regulation, and it is often difficult to determine both the genetic basis of adaptation and the evolutionary forces that influence regulation. In multiple evolution experiments with the bacteriophage ?X174, adaptive substitutions in cis-acting regulatory sequences sweep through the phage population as the result of strong positive selection at high temperatures that are non-permissive for laboratory-adapted phage. For one cis-regulatory region, we investigate the individual effects of four adaptive substitutions on transcript levels and fitness for phage growing on three hosts at two temperatures.The effect of the four individual substitutions on transcript levels is to down-regulate gene expression, regardless of temperature or host. To ascertain the conditions under which these substitutions are adaptive, fitness was measured by a variety of methods for several bacterial hosts growing at two temperatures, the control temperature of 37°C and the selective temperature of 42°C. Time to lysis and doublings per hour indicate that the four substitutions individually improve fitness over the ancestral strain at high temperature independent of the bacterial host in which the fitness was measured. Competition assays between the ancestral strain and either of two mutant strains indicate that both mutants out-compete the ancestor at high temperature, but the relative frequencies of each phage remain the same at the control temperature.Our results strongly suggest that gene transcription plays an important role in influencing fitness in the bacteriophage ?X174, and different point mutations in a single cis-regulatory region provided the genetic basis for this role in adaptation to high temperature. We speculate that the adaptive nature of these substitutions is due to the physiology of the host at high temperature or the need to maintain particular ratios of phage proteins during capsid assembly. Our investigation of regulatory evolution contributes to interpreting genome-level assessments of regulatory variation, as well as to understanding the molecular basis of adaptation.
| S-EPMC3003669 | biostudies-literature
Project description:The inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C is promoted by multivalent metal ions and by polyamines. The effect of the two types of cation is similar, and the concentration causing 50% inactivation varies inversely with the charge on the cation, although quadrivalent amines are less active than expected. The increase in activity as the charge rises suggests that electrostatic binding is overwhelmingly important.
| S-EPMC1152814 | biostudies-other
Project description:The need for Ca2+ in the inactivation of bacteriophage phi X174 by lipopolysaccharide from Escherichia coli C was confirmed. Ca2+ could be replaced almost completely by Na+, but the concentration of Na+ needed was greater by more than an order of magnitude. Other bivalent ions caused inactivation in the same way as Ca2+, and the degree of inactivation varied according to the ion. At 50% inactivation of bacteriophage, the relation between the concentrations of NaCl and of bivalent or tervalent ions (Mx+) fitted the conception that NaCl was neutralizing electrostatic repulsion between virus and lipopolysaccharide by an ionic-strength effect: that is, log[Mx+] varies inversely with square root[NaCl]. The variation in effect of bi- and ter-valent ions and the low concentration needed show that this is not an ionic-strength effect but likely to involve binding to more than one site.
| S-EPMC1144259 | biostudies-other
Project description:The DNA phage ?X174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides.
| S-EPMC3429702 | biostudies-other
Project description:In the present study, Z-DNA d(CGCGCG)2 was crystallized from a DNA solution in the absence of divalent metal cations and polyamines, and its X-ray structure was determined at 0.98 Å resolution. Comparison of this structure and previously reported Z-DNA structures, containing Mg(2+) cations and/or polyamines, demonstrated that Z-DNA can have structural fluctuations with respect to phosphate groups and hydration in the minor groove. At the GpC steps, a two-state structural equilibrium between the ZI and ZII conformations was frequently observed. In contrast, at the CpG steps, the phosphate groups exhibited rotational fluctuation, which could induce distortion of sugar puckering. In addition, alternative positions of water molecules were found in the middle of the minor groove of the Z-DNA. These structural fluctuations were likely observable because of the absence of Mg(2+) cations and polyamines. The results related to these phenomena were supported by those of other experimental methods, suggesting the possibility of these fluctuations occurring in biological conditions.
| S-EPMC3795545 | biostudies-literature
Project description:Viral mutation rates vary widely in nature, yet the mechanistic and evolutionary determinants of this variability remain unclear. Small DNA viruses mutate orders of magnitude faster than their hosts despite using host-encoded polymerases for replication, which suggests these viruses may avoid post-replicative repair. Supporting this, the genome of bacteriophage ?X174 is completely devoid of GATC sequence motifs, which are required for methyl-directed mismatch repair in Escherichia coli. Here, we show that restoration of the randomly expected number of GATC sites leads to an eightfold reduction in the rate of spontaneous mutation of the phage, without severely impairing its replicative capacity over the short term. However, the efficacy of mismatch repair in the presence of GATC sites is limited by inefficient methylation of the viral DNA. Therefore, both GATC avoidance and DNA under-methylation elevate the mutation rate of the phage relative to that of the host. We also found that the effects of GATC sites on the phage mutation rate vary extensively depending on their specific location within the phage genome. Finally, the mutation rate reduction afforded by GATC sites is fully reverted under stress conditions, which up-regulate repair pathways and expression of error-prone host polymerases such as heat and treatment with the base analog 5-fluorouracil, suggesting that access to repair renders the phage sensitive to stress-induced mutagenesis.
| S-EPMC5014478 | biostudies-literature
Project description: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 ?X174 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.
| S-EPMC5974221 | biostudies-literature
Project description:Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ?X174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ?X174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ?X174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS.
| S-EPMC5748221 | biostudies-literature
Project description:To investigate how high temperature affects viral transcription, the absolute amounts of mRNA for six bacteriophage ?X174 genes were compared at 37 °C and 42 °C using Q-PCR. At 37 °C, mRNA levels for all genes were consistent with previous studies, but at 42 °C mRNA levels for four genes were significantly different from levels at 37 °C. Transcript levels were higher for genes B and D; the promoter before gene B appears to be up-regulated at high temperature. Levels for genes F and G were reduced at high temperature, possibly due to increased efficiency of the transcription termination signal immediately upstream of gene F. These functional changes in ?X174 gene regulation at high temperature have not been described previously. Studies of phage evolution at high temperatures indicate that this difference in transcript levels is subject to adaptation.
| S-EPMC3335065 | biostudies-literature