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Viruses Sep 2021An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses,... (Review)
Review
An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.
Topics: Alphaherpesvirinae; Animals; Betaherpesvirinae; Dinucleoside Phosphates; Evolution, Molecular; Gammaherpesvirinae; Gene Expression; Genome, Viral; Herpesviridae; Host-Pathogen Interactions; Humans; Interferons; RNA Splicing; RNA, Viral; RNA-Binding Proteins; Signal Transduction; Viral Proteins
PubMed: 34578438
DOI: 10.3390/v13091857 -
Journal of the American Chemical Society Oct 2021Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the...
Triazole linkages (TLs) are mimics of the phosphodiester bond in oligonucleotides with applications in synthetic biology and biotechnology. Here we report the RuAAC-catalyzed synthesis of a novel 1,5-disubstituted triazole (TL) dinucleoside phosphoramidite as well as its incorporation into oligonucleotides and compare its DNA polymerase replication competency with other TL analogues. We demonstrate that TL has superior replication kinetics to these analogues and is accurately replicated by polymerases. Derived structure-biocompatibility relationships show that linker length and the orientation of a hydrogen bond acceptor are critical and provide further guidance for the rational design of artificial biocompatible nucleic acid backbones.
Topics: Catalysis; DNA; DNA-Directed DNA Polymerase; Dinucleoside Phosphates; Molecular Mimicry; Triazoles
PubMed: 34546729
DOI: 10.1021/jacs.1c08057 -
ELife Sep 2021Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network...
Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in . Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.
Topics: Bacterial Proteins; Binding Sites; Biofilms; Catalysis; Dinucleoside Phosphates; Enzyme Activation; Escherichia coli Proteins; Phosphorus-Oxygen Lyases; Protein Binding; Protein Conformation; Pseudomonas aeruginosa; Signal Transduction; Structure-Activity Relationship
PubMed: 34498587
DOI: 10.7554/eLife.67289 -
PLoS Biology Sep 2021Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host...
Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.
Topics: A549 Cells; Cell Line; Dinucleoside Phosphates; Humans; Interferon Regulatory Factors; Interferon-beta; RNA, Messenger; RNA, Viral; RNA-Binding Proteins; Virus Physiological Phenomena; Viruses
PubMed: 34491982
DOI: 10.1371/journal.pbio.3001352 -
Analytical and Bioanalytical Chemistry Nov 2021Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from...
Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2'3' cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3'3' cGAMP, 2'3' cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings. Graphical abstract.
Topics: Animals; Chromatography, Liquid; Cyclic GMP; Dinucleoside Phosphates; Escherichia coli; Fundulidae; Nucleotides, Cyclic; Tandem Mass Spectrometry
PubMed: 34476522
DOI: 10.1007/s00216-021-03628-6 -
Nucleic Acids Research Sep 2021The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with...
The cyclic dinucleotide second messenger c-di-AMP is a major player in regulation of potassium homeostasis and osmolyte transport in a variety of bacteria. Along with various direct interactions with proteins such as potassium channels, the second messenger also specifically binds to transcription factors, thereby altering the processes in the cell on the transcriptional level. We here describe the structural and biochemical characterization of BusR from the human pathogen Streptococcus agalactiae. BusR is a member of a yet structurally uncharacterized subfamily of the GntR family of transcription factors that downregulates transcription of the genes for the BusA (OpuA) glycine-betaine transporter upon c-di-AMP binding. We report crystal structures of full-length BusR, its apo and c-di-AMP bound effector domain, as well as cryo-EM structures of BusR bound to its operator DNA. Our structural data, supported by biochemical and biophysical data, reveal that BusR utilizes a unique domain assembly with a tetrameric coiled-coil in between the binding platforms, serving as a molecular ruler to specifically recognize a 22 bp separated bipartite binding motif. Binding of c-di-AMP to BusR induces a shift in equilibrium from an inactivated towards an activated state that allows BusR to bind the target DNA, leading to transcriptional repression.
Topics: ATP-Binding Cassette Transporters; Biological Transport; Crystallography, X-Ray; DNA, Bacterial; DNA-Binding Proteins; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Potassium; Protein Domains; Second Messenger Systems; Streptococcus agalactiae; Transcription Factors
PubMed: 34432045
DOI: 10.1093/nar/gkab736 -
Infection and Immunity Oct 2021Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial...
Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis () and degradation ( and ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ strain) or c-di-AMP accumulation (Δ, Δ, and Δ Δ strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the Δ strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine , and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the Δ strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both and conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.
Topics: Animals; Biofilms; Dinucleoside Phosphates; Enterococcus faecalis; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Gram-Positive Bacterial Infections; Humans; Virulence
PubMed: 34424750
DOI: 10.1128/IAI.00365-21 -
Biochimica Et Biophysica Acta. General... Nov 2021Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional...
Biochemical, crystallographic and biophysical characterization of histidine triad nucleotide-binding protein 2 with different ligands including a non-hydrolyzable analog of Ap4A.
BACKGROUND
Human HINT2 is an important mitochondrial enzyme involved in many processes such as apoptosis and bioenergetics, but its endogenous substrates and the three-dimensional structure of the full-length protein have not been identified yet.
METHODS
An HPLC assay was used to test the hydrolytic activity of HINT2 against various adenosine, guanosine, and 2'-deoxyguanosine derivatives containing phosphate bonds of different types and different leaving groups. Data on binding affinity were obtained by microscale thermophoresis (MST). Crystal structures of HINT2, in its apo form and with a dGMP ligand, were resolved to atomic resolution.
RESULTS
HINT2 substrate specificity was similar to that of HINT1, but with the major exception of remarkable discrimination against substrates lacking the 2'-hydroxyl group. The biochemical results were consistent with binding affinity measurements. They showed a similar binding strength of AMP and GMP to HINT2, and much weaker binding of dGMP, in contrast to HINT1. A non-hydrolyzable analog of Ap4A (JB419) interacted with both proteins with similar K and Ap4A is the signaling molecule that can interact with hHINT1 and regulate the activity of some transcription factors.
CONCLUSIONS
Several forms of homo- and heterodimers of different lengths of N-terminally truncated polypeptides resulting from degradation of the full-length protein were described. Ser144 in HINT2 appeared to be functionally equivalent to Ser107 in HINT1 by supporting the protonation of the leaving group in the hydrolytic mechanism of HINT2.
SIGNIFICANCE
Our results should be considered in future studies on the natural function of HINT2 and its role in nucleotide prodrug processing.
Topics: Dinucleoside Phosphates; Humans; Ligands; Mitochondrial Proteins
PubMed: 34329705
DOI: 10.1016/j.bbagen.2021.129968 -
Journal of Bacteriology Sep 20213'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and...
3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.
Topics: Bacillus subtilis; Bacterial Proteins; Biosensing Techniques; Dinucleoside Phosphates; Fluorescence Resonance Energy Transfer; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Models, Molecular; Mutation; Nucleotides; Protein Conformation
PubMed: 34309402
DOI: 10.1128/JB.00080-21 -
Nucleic Acids Research Sep 2021The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide....
The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.
Topics: Binding Sites; DNA Methylation; DNA-Binding Proteins; Dinucleoside Phosphates; Evolution, Molecular; Genome, Human; Humans; Nucleic Acid Conformation; Receptors, Glucocorticoid; Regulatory Sequences, Nucleic Acid; Thymine
PubMed: 34289059
DOI: 10.1093/nar/gkab605