Genome wide-scale CRISPR-Cas9 knockout screens identify a fitness score for optimized risk stratification in colorectal cancer.

BACKGROUND: The molecular complexity of colorectal cancer poses a significant challenge to the clinical implementation of accurate risk stratification. There is still an urgent need to find better biomarkers to enhance established risk stratification and guide risk-adapted treatment decisions. METHODS: we systematically analyzed cancer dependencies of 17 colorectal cancer cells and 513 other cancer cells based on genome-scale CRISPR-Cas9 knockout screens to identify colorectal cancer-specific fitness genes. A regression model was built using colorectal cancer-specific fitness genes, which was validated in other three independent cohorts. 30 published gene expression signatures were also retrieved. FINDINGS: We defined a total of 1828 genes that were colorectal cancer-specific fitness genes and identified a 22 colorectal cancer-specific fitness gene (CFG22) score. A high CFG22 score represented unfavorable recurrence and mortality rates, which was validated in three independent cohorts. Combined with age, and TNM stage, the CFG22 model can provide guidance for the prognosis of colorectal cancer patients. Analysis of genomic abnormalities and infiltrating immune cells in the CFG22 risk stratification revealed molecular pathological difference between the subgroups. Besides, drug analysis found that CFG22 high patients were more sensitive to clofibrate. INTERPRETATION: The CFG22 model provided a powerful auxiliary prediction tool for identifying colorectal cancer patients with high recurrence risk and poor prognosis, optimizing precise treatment and improving clinical efficacy.

Elevated [CO2] and temperature augment gas exchange and shift the fitness landscape in a montane forb.

Climate change is simultaneously increasing carbon dioxide concentrations ([CO2]) and temperature. These factors could interact to influence plant physiology and performance. Alternatively, increased [CO2] may offset costs associated with elevated temperatures. Furthermore, the interaction between elevated temperature and [CO2] may differentially affect populations from along an elevational gradient and disrupt local adaptation. We conducted a multifactorial growth chamber experiment to examine the interactive effects of temperature and [CO2] on fitness and ecophysiology of diverse accessions of Boechera stricta (Brassicaceae) sourced from a broad elevational gradient in Colorado. We tested whether increased [CO2] would enhance photosynthesis across accessions, and whether warmer conditions would depress the fitness of high-elevation accessions owing to steep reductions in temperature with increasing elevation in this system. Elevational clines in [CO2] are not as evident, making it challenging to predict how locally adapted ecotypes will respond to elevated [CO2]. This experiment revealed that elevated [CO2] increased photosynthesis and intrinsic water use efficiency across all accessions. However, these instantaneous responses to treatments did not translate to changes in fitness. Instead, increased temperatures reduced the probability of reproduction for all accessions. Elevated [CO2] and increased temperatures interacted to shift the adaptive landscape, favoring lower elevation accessions for the probability of survival and fecundity. Our results suggest that elevated temperatures and [CO2] associated with climate change could have severe negative consequences, especially for high-elevation populations.

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

Variabilidad genética del pez Lutjanus guttatus (Perciformes: Lutjanidae) mediante el uso de microsatélites en Costa Rica / Genetic variability of the fish Lutjanus guttatus (Perciformes: Lutjanidae) using microsatellites in Costa Rica

Introducción: El pargo mancha es un pez marino de alto consumo e interés comercial en Costa Rica que está sometido a una fuerte presión pesquera, la cual puede afectar la diversidad genética y generar problemas por depresión endogámica. Objetivo: Evaluar el estado genético de la población de Lutjanus guttatus mediante el uso microsatélites. Métodos: Se recolectaron muestras entre el 2018 y 2019 y se estudiaron 44 individuos de cada una de las localidades del Golfo de Nicoya y Golfo Dulce. Se realizó la extracción de ADN y la amplificación de diez loci con microsatélites mediante PCR, para la determinación del genotipo, análisis de diversidad genética y estructura poblacional. Resultados: Los parámetros de diversidad indican un elevado polimorfismo asociado con un alto número de alelos obtenidos por locus, pero con bajos niveles de heterocigosidad observada en comparación con la esperada (Ho= 0.774 y 0.800 y He= 0.948 y 0.954 para Golfo de Nicoya y Golfo Dulce, respectivamente). No hay evidencia suficiente para decir que las dos poblaciones son distintas (FST= 0.00264, P > 0.05). La desviación del Equilibrio de Hardy-Weinberg indica la posible mezcla de organismos de origen distinto a los del medio silvestre. Conclusiones: L. guttatus tiene niveles altos de diversidad genética, no hay evidencia de diferenciación en subpoblaciones genéticas, lo que en manejo pesquerías se considera una sola población panmíctica. La posible mezcla de individuos de origen distinto al silvestre sugiere la presencia de organismos de un programa de repoblación o de cultivos comerciales en la región. El uso de marcadores genéticos se recomienda para el monitoreo, además, en programas de repoblación y evaluar su efecto.

Introduction: The spotted snapper is a high-consumption and commercially important marine fish in Costa Rica, subjected to heavy fishing pressures, which can affect genetic diversity and generate problems due to inbreeding depression. Objective: To evaluate the genetic status of the population of Lutjanus guttatus using microsatellites. Methods: Samples were collected between 2018 and 2019, and 44 individuals from each of the localities of the Gulf of Nicoya and the Gulf of Dulce were studied. DNA extraction and amplification of ten loci with microsatellites using PCR were performed, followed by genotyping, analysis of genetic diversity, and population structure. Results: Diversity parameters indicate a high polymorphism associated with a high number of alleles obtained per locus, but with low levels of observed heterozygosity compared to expected (Ho= 0.774 and 0.800, and He= 0.948 and 0.954 for the Gulf of Nicoya and Gulf of Dulce, respectively). There is not enough evidence to say that the two populations are distinct (FST= 0.00264, P > 0.05). Deviation from Hardy-Weinberg equilibrium was recorded, indicating possible mixing of organisms of different origin from the wild environment. Conclusions: L. guttatus presents high levels of genetic diversity, without evidence of differentiation in genetic subpopulations. For fisheries management purposes, they would be considered a single panmictic population. The possible mixing with wild individuals suggests the presence of organisms derived from a restocking or commercial cultivation program carried out in the region. The use of genetic markers is recommended to maintain monitoring, follow up on restocking programs and evaluate their effect.

Nanoplastic exposure in soil compromises the energy budget of the soil nematode C. elegans and decreases reproductive fitness.

Environmental nanoplastics (NPs) can accumulate in soils, posing a potential risk to soil ecosystems. However, the ecotoxicity of NPs for soil organisms has received little research attention. This study investigated whether NP exposure in soil leads to reproductive decline in the soil nematode Caenorhabditis elegans and sought to determine the mechanisms by which it may occur. Wild-type N2 C. elegans L1 larvae were exposed to various concentrations of nano-sized polystyrene (100 nm) in soil (0, 1, 10, 100, and 1000 mg/kg dry weight) for 96 h. We show that nano-sized polystyrene (100 nm) labeled with red fluorescence significantly accumulated in the intestine of C. elegans in a dose-dependent fashion via soil exposure (8%-47% increase). In addition, NP soil exposure led to 7%-33% decline in the number of eggs in utero and 2.6%-4.4% decline in the egg hatching percentage. We also find that the number of germ cell corpses (31%-55% increase) and the mRNA levels of germline apoptosis marker gene ced-3 (14%-31% increase) were significantly higher with greater NP soil exposure (10, 100, and 1000 mg/kg), while intracellular ATP levels were significantly reduced. Finally, the DEBtox model, which is based on the dynamic energy budget theory, was applied to show that the increased reproductive costs for C. elegans caused by NPs in soil are associated with energy depletion and reproductive decline. The threshold value (4.18 × 10-6 mg/kg) for the energy budget also highlighted the potential high reproductive risk posed by NPs in terrestrial ecosystems. Our study provides new insights into how soil organisms interact with NPs in soil ecosystems.

Adaptation of the yeast gene knockout collection is near-perfectly predicted by fitness and diminishing return epistasis.

Adaptive evolution of clonally dividing cells and microbes is the ultimate cause of cancer and infectious diseases. The possibility of constraining the adaptation of cell populations, by inhibiting proteins enhancing the evolvability, has therefore attracted interest. However, our current understanding of how genes influence adaptation kinetics is limited, partly because accurately measuring adaptation for many cell populations is challenging. We used a high-throughput adaptive laboratory evolution platform to track the adaptation of >18,000 cell populations corresponding to single-gene deletion strains in the haploid yeast deletion collection. We report that the preadaptation fitness of gene knockouts near-perfectly (R2= 0.91) predicts their adaptation to arsenic, leaving at the most a marginal role for dedicated evolvability gene functions. We tracked the adaptation of another >23,000 gene knockout populations to a diverse range of selection pressures and generalized the almost perfect (R2=0.72-0.98) capacity of preadaptation fitness to predict adaptation. We also reconstructed mutations in FPS1, ASK10, and ARR3, which together account for almost all arsenic adaptation in wild-type cells, in gene deletions covering a broad fitness range and show that the predictability of arsenic adaptation can be understood as a by global epistasis, where excluding arsenic is more beneficial to arsenic unfit cells. The paucity of genes with a meaningful evolvability effect on adaptation diminishes the prospects of developing adjuvant drugs aiming to slow antimicrobial and chemotherapy resistance.

Inferring Epistasis from Genetic Time-series Data.

Epistasis refers to fitness or functional effects of mutations that depend on the sequence background in which these mutations arise. Epistasis is prevalent in nature, including populations of viruses, bacteria, and cancers, and can contribute to the evolution of drug resistance and immune escape. However, it is difficult to directly estimate epistatic effects from sampled observations of a population. At present, there are very few methods that can disentangle the effects of selection (including epistasis), mutation, recombination, genetic drift, and genetic linkage in evolving populations. Here we develop a method to infer epistasis, along with the fitness effects of individual mutations, from observed evolutionary histories. Simulations show that we can accurately infer pairwise epistatic interactions provided that there is sufficient genetic diversity in the data. Our method also allows us to identify which fitness parameters can be reliably inferred from a particular data set and which ones are unidentifiable. Our approach therefore allows for the inference of more complex models of selection from time-series genetic data, while also quantifying uncertainty in the inferred parameters.

Proteolytic Processing of the Coronavirus Replicase Nonstructural Protein 14 Exonuclease Is Not Required for Virus Replication but Alters RNA Synthesis and Viral Fitness.

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.

Feline Fertility: Consequences of inbreeding and implications for reproductive fitness.

PRACTICAL RELEVANCE: When compared with the number of individuals that make up a dog breed, the population within a given cat breed is very small. Therefore, to maintain a breed standard, a certain degree of inbreeding is necessary. However, when inbreeding reaches a certain threshold, it can lead to decreased fertility, which manifests as failure to conceive, smaller litter size, increased neonatal illness and neonatal mortality. Breeders should be encouraged to keep comprehensive records on breeding outcomes, including number of kittens born, neonatal vitality, daily kitten weights and kitten health at weaning. Commercially available DNA panels are available to inform and facilitate excellent breeding choices and can estimate the coefficient of inbreeding. Clinicians should include a review of the degree of inbreeding in the work-up for any cattery or cat colony experiencing decreased fertility. AIM: The objective of this article is to provide clinicians, especially those working with cat breeders, with an easy-to-understand guide to genetics and to demonstrate how inbreeding influences fertility and neonatal survival. EQUIPMENT AND TECHNICAL SKILLS: Medical records and the pedigree of the cats in question are required to investigate cases of infertility that may be related to inbreeding. A DNA analysis kit that measures genetic diversity and health parameters can also be helpful; those that have been developed by geneticists and veterinarians at universities are preferable, as they include access to highly skilled genetic counselors and researchers who are open to working up newly discovered genetic diseases. EVIDENCE BASE: The material provided is based on current literature and the author's own studies examining outcomes in a closed cattery.

C16orf72/HAPSTR1 is a molecular rheostat in an integrated network of stress response pathways.

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.

Fundamental immune-oncogenicity trade-offs define driver mutation fitness.

Missense driver mutations in cancer are concentrated in a few hotspots1. Various mechanisms have been proposed to explain this skew, including biased mutational processes2, phenotypic differences3-6 and immunoediting of neoantigens7,8; however, to our knowledge, no existing model weighs the relative contribution of these features to tumour evolution. We propose a unified theoretical 'free fitness' framework that parsimoniously integrates multimodal genomic, epigenetic, transcriptomic and proteomic data into a biophysical model of the rate-limiting processes underlying the fitness advantage conferred on cancer cells by driver gene mutations. Focusing on TP53, the most mutated gene in cancer1, we present an inference of mutant p53 concentration and demonstrate that TP53 hotspot mutations optimally solve an evolutionary trade-off between oncogenic potential and neoantigen immunogenicity. Our model anticipates patient survival in The Cancer Genome Atlas and patients with lung cancer treated with immunotherapy as well as the age of tumour onset in germline carriers of TP53 variants. The predicted differential immunogenicity between hotspot mutations was validated experimentally in patients with cancer and in a unique large dataset of healthy individuals. Our data indicate that immune selective pressure on TP53 mutations has a smaller role in non-cancerous lesions than in tumours, suggesting that targeted immunotherapy may offer an early prophylactic opportunity for the former. Determining the relative contribution of immunogenicity and oncogenic function to the selective advantage of hotspot mutations thus has important implications for both precision immunotherapies and our understanding of tumour evolution.

Hybridization alters the shape of the genotypic fitness landscape, increasing access to novel fitness peaks during adaptive radiation.

Estimating the complex relationship between fitness and genotype or phenotype (i.e. the adaptive landscape) is one of the central goals of evolutionary biology. However, adaptive walks connecting genotypes to organismal fitness, speciation, and novel ecological niches are still poorly understood and processes for surmounting fitness valleys remain controversial. One outstanding system for addressing these connections is a recent adaptive radiation of ecologically and morphologically novel pupfishes (a generalist, molluscivore, and scale-eater) endemic to San Salvador Island, Bahamas. We leveraged whole-genome sequencing of 139 hybrids from two independent field fitness experiments to identify the genomic basis of fitness, estimate genotypic fitness networks, and measure the accessibility of adaptive walks on the fitness landscape. We identified 132 single nucleotide polymorphisms (SNPs) that were significantly associated with fitness in field enclosures. Six out of the 13 regions most strongly associated with fitness contained differentially expressed genes and fixed SNPs between trophic specialists; one gene (mettl21e) was also misexpressed in lab-reared hybrids, suggesting a potential intrinsic genetic incompatibility. We then constructed genotypic fitness networks from adaptive alleles and show that scale-eating specialists are the most isolated of the three species on these networks. Intriguingly, introgressed and de novo variants reduced fitness landscape ruggedness as compared to standing variation, increasing the accessibility of genotypic fitness paths from generalist to specialists. Our results suggest that adaptive introgression and de novo mutations alter the shape of the fitness landscape, providing key connections in adaptive walks circumventing fitness valleys and triggering the evolution of novelty during adaptive radiation.

One of the main drivers of evolution is natural selection, which is when organisms better adapted to their environment are more likely to survive and reproduce. A common metaphor to explain this process is a landscape covered in peaks and valleys: the peaks represent genetic combinations or traits with high evolutionary fitness, while the valleys represent those with low fitness. As a population evolves and its environment changes, it moves among these peaks taking small steps across the landscape. However, there is a limit to how far an organism can travel in one leap. So, what happens when they need to cross a valley of low fitness to get to the next peak? To address this question, Patton et al. studied three young species of pupfish that recently evolved from a common ancestor and co-habit the same environment in the Caribbean. Patton et al. sequenced whole genomes of each new species and used this to build a genotypic fitness landscape, a network linking neighboring genotypes which each have a unique fitness value that was measured during field experiments. This revealed that most of the paths connecting the different species passed through valleys of low fitness. But there were rare, narrow ridges connecting each species. Next, Patton et al. found that new mutations as well as genetic variations that arose from mating with pupfish on other Caribbean islands altered genetic interactions and changed the shape of the fitness landscape. Ultimately, this significantly increased the accessibility of fitness peaks by both adding more ridges and decreasing the lengths of paths, expanding the realm of possible evolutionary outcomes. Understanding how fitness landscapes change during evolution could help to explain where new species come from. Other researchers could apply the same approach to estimate the genotypic fitness landscapes of other species, from bacteria to vertebrates. These networks could be used to visualize the complex fitness landscape that connects all lifeforms on Earth.

Binucleation ramps up gene expression meeting the physiological demands of an organism.

In this issue of PLOS Biology, van Rijnberk and colleagues show how polyploidy, via binucleation, enables Caenorhabditis elegans intestinal cells to ramp up gene expression supplying the oocytes with the necessary lipids for optimal organismal growth and reproductive fitness.

A general calculus of fitness landscapes finds genes under selection in cancers.

Genetic variants drive the evolution of traits and diseases. We previously modeled these variants as small displacements in fitness landscapes and estimated their functional impact by differentiating the evolutionary relationship between genotype and phenotype. Conversely, here we integrate these derivatives to identify genes steering specific traits. Over cancer cohorts, integration identified 460 likely tumor-driving genes. Many have literature and experimental support but had eluded prior genomic searches for positive selection in tumors. Beyond providing cancer insights, these results introduce a general calculus of evolution to quantify the genotype-phenotype relationship and discover genes associated with complex traits and diseases.

Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

Genome instability drives epistatic adaptation in the human pathogen Leishmania.

How genome instability is harnessed for fitness gain despite its potential deleterious effects is largely elusive. An ideal system to address this important open question is provided by the protozoan pathogen Leishmania, which exploits frequent variations in chromosome and gene copy number to regulate expression levels. Using ecological genomics and experimental evolution approaches, we provide evidence that Leishmania adaptation relies on epistatic interactions between functionally associated gene copy number variations in pathways driving fitness gain in a given environment. We further uncover posttranscriptional regulation as a key mechanism that compensates for deleterious gene dosage effects and provides phenotypic robustness to genetically heterogenous parasite populations. Finally, we correlate dynamic variations in small nucleolar RNA (snoRNA) gene dosage with changes in ribosomal RNA 2'-O-methylation and pseudouridylation, suggesting translational control as an additional layer of parasite adaptation. Leishmania genome instability is thus harnessed for fitness gain by genome-dependent variations in gene expression and genome-independent compensatory mechanisms. This allows for polyclonal adaptation and maintenance of genetic heterogeneity despite strong selective pressure. The epistatic adaptation described here needs to be considered in Leishmania epidemiology and biomarker discovery and may be relevant to other fast-evolving eukaryotic cells that exploit genome instability for adaptation, such as fungal pathogens or cancer.

Genome-wide analysis of fitness-factors in uropathogenic Escherichia coli during growth in laboratory media and during urinary tract infections.

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

Nucleocapsid mutations R203K/G204R increase the infectivity, fitness, and virulence of SARS-CoV-2.

Previous work found that the co-occurring mutations R203K/G204R on the SARS-CoV-2 nucleocapsid (N) protein are increasing in frequency among emerging variants of concern or interest. Through a combination of in silico analyses, this study demonstrates that R203K/G204R are adaptive, while large-scale phylogenetic analyses indicate that R203K/G204R associate with the emergence of the high-transmissibility SARS-CoV-2 lineage B.1.1.7. Competition experiments suggest that the 203K/204R variants possess a replication advantage over the preceding R203/G204 variants, possibly related to ribonucleocapsid (RNP) assembly. Moreover, the 203K/204R virus shows increased infectivity in human lung cells and hamsters. Accordingly, we observe a positive association between increased COVID-19 severity and sample frequency of 203K/204R. Our work suggests that the 203K/204R mutations contribute to the increased transmission and virulence of select SARS-CoV-2 variants. In addition to mutations in the spike protein, mutations in the nucleocapsid protein are important for viral spreading during the pandemic.

The biology of papillomavirus PDZ associations: what do they offer papillomaviruses?

The high-risk α-type papillomaviruses have a C-terminal PDZ-binding motif (PBM) on one of the two major oncoproteins E6 or E7; the vast majority on E6. The PBM is essential for the high-risk HPV life cycle, for episomal maintenance of the virus genome, and for maintaining the mitotic stability of the infected cell. The question is why only these viruses have PBMs - are there specific constraints imposed by the mucosal epithelium in which these viruses replicate? However the low-risk α-HPVs, such as HPV-6 and HPV-11 replicate extremely efficiently without a PBM, while viruses of the alpha8 group, such as HPV-40, replicate well with a very primitive PBM. So what does PDZ-binding capacity contribute to the fitness of the virus?