RNA viruses exist as genetically diverse populations. It is thought that diversity and genetic structure of viral populations determine the rapid adaptation observed in RNA viruses and hence their ...pathogenesis. However, our understanding of the mechanisms underlying virus evolution has been limited by the inability to accurately describe the genetic structure of virus populations. Next-generation sequencing technologies generate data of sufficient depth to characterize virus populations, but are limited in their utility because most variants are present at very low frequencies and are thus indistinguishable from next-generation sequencing errors. Here we present an approach that reduces next-generation sequencing errors and allows the description of virus populations with unprecedented accuracy. Using this approach, we define the mutation rates of poliovirus and uncover the mutation landscape of the population. Furthermore, by monitoring changes in variant frequencies on serially passaged populations, we determined fitness values for thousands of mutations across the viral genome. Mapping of these fitness values onto three-dimensional structures of viral proteins offers a powerful approach for exploring structure-function relationships and potentially uncovering new functions. To our knowledge, our study provides the first single-nucleotide fitness landscape of an evolving RNA virus and establishes a general experimental platform for studying the genetic changes underlying the evolution of virus populations.
Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses ...to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.
Large parts of mammalian genomes are transcriptionally inactive and enriched with various classes of interspersed and tandem repeats. Here we show that the tumor suppressor protein p53 cooperates ...with DNA methylation to maintain silencing of a large portion of the mouse genome. Massive transcription of major classes of short, interspersed nuclear elements (SINEs) B1 and B2, both strands of near-centromeric satellite DNAs consisting of tandem repeats, and multiple species of noncoding RNAs was observed in p53-deficient but not in p53 wild-type mouse fibroblasts treated with the DNA demethylating agent 5-aza-2’-deoxycytidine. The abundance of these transcripts exceeded the level of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming double-stranded RNA (dsRNA), was accompanied by a strong, endogenous, apoptosis-inducing type I IFN response. This phenomenon, which we named “TRAIN” (for “transcription of repeats activates interferon”), was observed in spontaneous tumors in two models of cancer-prone mice, presumably reflecting naturally occurring DNA hypomethylation and p53 inactivation in cancer. These observations suggest that p53 and IFN cooperate to prevent accumulation of cells with activated repeats and provide a plausible explanation for the deregulation of IFN function frequently seen in tumors. Overall, this work reveals roles for p53 and IFN that are key for genetic stability and therefore relevant to both tumorigenesis and the evolution of species.
RNA viruses, such as poliovirus, have a great evolutionary capacity, allowing them to quickly adapt and overcome challenges encountered during infection. Here we show that poliovirus infection in ...immune-competent mice requires adaptation to tissue-specific innate immune microenvironments. The ability of the virus to establish robust infection and virulence correlates with its evolutionary capacity. We further identify a region in the multi-functional poliovirus protein 2B as a hotspot for the accumulation of minor alleles that facilitate a more effective suppression of the interferon response. We propose that population genetic dynamics enables poliovirus spread between tissues through optimization of the genetic composition of low frequency variants, which together cooperate to circumvent tissue-specific challenges. Thus, intrahost virus evolution determines pathogenesis, allowing a dynamic regulation of viral functions required to overcome barriers to infection.RNA viruses, such as polioviruses, have a great evolutionary capacity and can adapt quickly during infection. Here, the authors show that poliovirus infection in mice requires adaptation to innate immune microenvironments encountered in different tissues.
Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different ...genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations.
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•Recombination accelerates adaptation in the short time frame of acute poliovirus infection•Recombination is essential to enrich beneficial mutations and purge deleterious mutations•Virus adapts most rapidly at a mutation rate set by trade-off of selection and harmful mutations
Mutation and recombination drive evolution, but whether these processes act concertedly remains unclear. Xiao et al. demonstrate that recombination accelerates poliovirus adaptation and evolution. Recombination enriches beneficial mutations while purging deleterious mutations. Poliovirus adapts most rapidly at an optimal mutation rate determined by trade-off between selection and accumulation of detrimental mutations.
Transitions of B-DNA to alternative DNA structures (ADS) can be triggered by negative torsional strain, which occurs during replication and transcription, and may lead to genomic instability. ...However, how ADS are recognized in cells is unclear. We found that the binding of candidate anticancer drug, curaxin, to cellular DNA results in uncoiling of nucleosomal DNA, accumulation of negative supercoiling and conversion of multiple regions of genomic DNA into left-handed Z-form. Histone chaperone FACT binds rapidly to the same regions via the SSRP1 subunit in curaxin-treated cells. In vitro binding of purified SSRP1 or its isolated CID domain to a methylated DNA fragment containing alternating purine/pyrimidines, which is prone to Z-DNA transition, is much stronger than to other types of DNA. We propose that FACT can recognize and bind Z-DNA or DNA in transition from a B to Z form. Binding of FACT to these genomic regions triggers a p53 response. Furthermore, FACT has been shown to bind to other types of ADS through a different structural domain, which also leads to p53 activation. Thus, we propose that FACT acts as a sensor of ADS formation in cells. Recognition of ADS by FACT followed by a p53 response may explain the role of FACT in DNA damage prevention.
Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate
enter torpor in response to changes in ...seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of
microbiome and its functionality in active and dormant colonies, using microscopy, qPCR,
hybridization, genomics and transcriptomics. A novel lineage of
, proposed here as
Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that
can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host,
, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.
Metabolite composition offers a powerful tool for understanding gene function and regulatory processes. However, metabolomics studies on multicellular organisms have thus far been performed primarily ...on whole organisms, organs, or cell lines, losing information about individual cell types within a tissue. With the goal of profiling metabolite content in different cell populations within an organ, we used FACS to dissect GFP-marked cells from Arabidopsis roots for metabolomics analysis. Here, we present the metabolic profiles obtained from five GFP-tagged lines representing core cell types in the root. Fifty metabolites were putatively identified, with the most prominent groups being glucosinolates, phenylpropanoids, and dipeptides, the latter of which is not yet explored in roots. The mRNA expression of enzymes or regulators in the corresponding biosynthetic pathways was compared with the relative metabolite abundance. Positive correlations suggest that the rate-limiting steps in biosynthesis of glucosinolates in the root are oxidative modifications of side chains. The current study presents a work flow for metabolomics analyses of cell-type populations.
The output of LC-MS metabolomics experiments consists of mass-peak intensities identified through a peak-picking/alignment procedure. Besides imperfections in biological samples and instrumentation, ...data accuracy is highly dependent on the applied algorithms and their parameters. Consequently, quality control (QC) is essential for further data analysis. Here, we present a QC approach that is based on discrepancies between replicate samples. First, the quantile normalization of per-sample log-signal distributions is applied to each group of biologically homogeneous samples. Next, the overall quality of each replicate group is characterized by the Z-transformed correlation coefficients between samples. This general QC allows a tuning of the procedure's parameters which minimizes the inter-replicate discrepancies in the generated output. Subsequently, an in-depth QC measure detects local neighborhoods on a template of aligned chromatograms that are enriched by divergences between intensity profiles of replicate samples. These neighborhoods are determined through a segmentation algorithm. The retention time (RT)-m/z positions of the neighborhoods with local divergences are indicative of either: incorrect alignment of chromatographic features, technical problems in the chromatograms, or to a true biological discrepancy between replicates for particular metabolites. We expect this method to aid in the accurate analysis of metabolomics data and in the development of new peak-picking/alignment procedures.
Summary Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The ...actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor α (ERα) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERα and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter.
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