Biology subjects

Lu, X.

Publications and source records attributed to Lu, X..

At least 361 records · Page 20Linked to original sources

bioRxiv2018

Kinase Driver Mutations in Protein-protein Structure may associate to Disease by Effecting Kinase Stability

Activating mutations are significant drug targets of diseases. Statistical analysis on the mutated amino acids, the mutation characteristic and the related disease information of activating mutations is of great significance for the diagnosis and treatment of diseases. Study on the protein stability by predicting Gibbs free energy ({Delta}{Delta}G) change after mutation is very helpful for understanding the relationship between protein structure and function. By combining mCSM and disease datasets, this paper studies the effect of mutation on the protein stability and disease. The results show that the mutations in protein may be the cause of disease for PPI structures, which statistically afford significant information for disease related research and medical diagnosis and treatment.

biochemistry↗

bioRxiv2018

Regulatory networks of gene expression in maize (Zea mays) under drought stress and re-watering

Drought can severely limit plant growth and production. However, few studies have investigated gene expression profiles in maize during drought/re-watering. We compared drought-treated and water-sufficient maize plants by measuring their leaf relative water content, superoxide dismutase and peroxidase activities, proline content, and leaf gas exchange parameters (photosynthetic rates, stomatal conductance, and transpiration rates). We conducted RNA sequencing analyses to elucidate gene expression profiles and identify miRNAs that might be related to drought resistance. A GO enrichment analysis showed that the common DEGs (differently expressed genes) between drought-treated and control plants were involved in response to stimulus, cellular process, metabolic process, cell part, and binding and catalytic activity. Analyses of gene expression profiles revealed that 26 of the DEGs under drought encoded 10 enzymes involved in proline synthesis, suggesting that increased proline synthesis was a key part of the drought response. We also investigated cell wall-related genes and transcription factors regulating abscisic acid-dependent and -independent pathways. The expression profiles of the miRNAs miR6214-3p, miR5072-3p, zma-miR529-5p, zma-miR167e-5p, zma-miR167f-5p, and zma-miR167j-5p and their relevant targets under drought conditions were analyzed. These results provide new insights into the molecular mechanisms of drought tolerance, and may identify new targets for breeding drought-tolerant maize lines.\n\nAbbreviationsleaf relative water content: RWC, superoxide dismutase activity: SOD, peroxidase activity: POD, proline content: Pro, photosynthetic rates: Pn, stomatal conductance: Cond, transpiration rates: Tr.; quantitative real-time polymerase chain reaction: qPCR; abscisic acid; ABA; polyethylene glycol :PEG; Principal component analysis :PCA; polyacrylamide gel electrophoresis :PAGE\n\nHighlightThe study of physiology and molecular mechanism of maize laid a theoretical foundation for drought resistance breeding under drought stress and re-watering.

genomics↗

bioRxiv2018

Genetic, inflammatory, and tissue-specific factors control expression of human calpain-14

Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14. Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ~10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens mediated CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.

genomics↗

bioRxiv2018

Systematic Discovery of the Functional Impact of Somatic Genome Alterations in Individual Tumors through Tumor-specific Causal Inference

We report a tumor-specific causal inference (TCI) framework, which discovers causative somatic genome alterations (SGAs) through inferring causal relationships between SGAs and molecular phenotypes (e.g., transcriptomic, proteomic, or metabolomic changes) within an individual tumor. We applied the TCI algorithm to tumors from The Cancer Genome Atlas (TCGA) and identified those SGAs that causally regulate the differentially expressed genes (DEGs) within each tumor. Overall, TCI identified 634 SGAs that cause cancer-related DEGs in a significant number of tumors, including most of the previously known drivers and many novel candidate cancer drivers. The inferred causal relationships are statistically robust and biologically sensible, and multiple lines of experimental evidence support the predicted functional impact of both well-known and novel candidate drivers. By identifying major candidate drivers and revealing their functional impact in a tumor, TCI shed light on disease mechanisms of each tumor, providing useful information for advancing cancer biology and precision oncology.\n\nSignificance statementsCancer is mainly caused by SGAs. Precision oncology involves identifying and targeting tumor-specific aberrations resulting from causative SGAs. TCI is a novel computational framework for discovering the causative SGAs and their impact on oncogenic processes, thus revealing tumor-specific disease mechanisms. This information can be used to guide precision oncology.

cancer biology↗

bioRxiv2018

The Carcinogenome Project: In-vitro Gene Expression Profiling of Chemical Perturbations to Predict Long-Term Carcinogenicity

Background: Most chemicals in commerce have not been evaluated for their carcinogenic potential. The current de-facto gold-standard approach to carcinogen testing adopts the two-year rodent bioassay, a time consuming and costly procedure. Alternative approaches, such as high-throughput in-vitro assays, show promise in addressing the limitations in carcinogen screening.\n\nObjectives: We developed a screening process for predicting chemical carcinogenicity and genotoxicity and characterizing modes of actions (MoAs) using in-vitro gene expression assays.\n\nMethods: We generated a large toxicogenomics resource comprising ~6,000 expression profiles corresponding to 330 chemicals profiled in HepG2 cells at multiple doses and in replicates. Predictive models of carcinogenicity were built using a Random Forest classifier. Differential pathway enrichment analysis was performed to identify pathways associated with carcinogen exposure. Signatures of carcinogenicity and genotoxicity were compared with external data sources including Drugmatrix and the Connectivity Map.\n\nResults: Among profiles with sufficient bioactivity, our classifiers achieved 72.2% AUC for predicting carcinogenicity and 82.3% AUC for predicting genotoxicity. Our analysis showed that chemical bioactivity, as measured by the strength and reproducibility of the transcriptional response, is not significantly associated with long-term carcinogenicity, as evidenced by the many carcinogenic chemicals that did not elicit substantial changes in gene expression at doses up to 40 M. However, sufficiently high transcriptional bioactivity is necessary for a chemical to be used for prediction of carcinogenicity. Pathway enrichment analysis revealed several pathways consistent with literature review of pathways that drive cancer, including DNA damage and DNA repair. These data are available for download via https://clue.io/CRCGN_ABC, and a web portal for interactive query and visualization of the data and results is accessible at https://carcinogenome.org.\n\nConclusions: We demonstrated a short-term in-vitro screening approach using gene expression profiling to predict long-term carcinogenicity and infer MoAs of chemical perturbations.

bioinformatics↗

bioRxiv2018

Par3 regulates Rac1 signaling and microtubule organization during planar polarization of auditory hair cells

In the inner ear sensory epithelia, hair bundles atop sensory hair cells are mechanosensory apparati with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 plays a key role in planar polarization of hair cells. Par3 deletion in the inner ear resulted in defects in cochlear length, hair bundle orientation and kinocilium positioning. During PCP establishment, Par3 promotes localized Rac-Pak signaling through an interaction with Tiam1. Par3 regulates microtubule dynamics and organization, which is crucial for basal body positioning. Moreover, there is reciprocal regulation of Par3 and the core PCP molecule Vangl2. Thus, we conclude that Par3 is an effector and integrator of cell-intrinsic and tissue-level PCP signaling.\n\nOne sentence summaryPar3 regulates planar polarity of auditory hair cells

developmental biology↗

bioRxiv2018

Single cell RNA-seq reveals profound transcriptional similarity between Barretts esophagus and esophageal glands

Barretts esophagus is a precursor of esophageal adenocarcinoma. In this common condition, squamous epithelium in the esophagus is replaced by columnar epithelium in response to acid reflux. Barretts esophagus is highly heterogeneous and its relationships to normal tissues are unclear. We investigated the cellular complexity of Barretts esophagus and the upper gastrointestinal tract using RNA-sequencing of 2895 single cells from multiple biopsies from four patients with Barretts esophagus and two patients without esophageal pathology. We found that uncharacterised cell populations in Barretts esophagus, marked by LEFTY1 and OLFM4, exhibit a profound transcriptional overlap with a subset of esophageal cells, but not with gastric or duodenal cells. Additionally, SPINK4 and ITLN1 mark cells that precede morphologically identifiable goblet cells in colon and Barretts esophagus, potentially aiding the identification of metaplasia. Our findings reveal striking transcriptional relationships between normal tissue populations and cells in a premalignant condition, with implications for clinical practice.

genetics↗

bioRxiv2018

Sapropterin Treatment Prevents Congenital Heart Defects Induced by Pregestational Diabetes in Mice

AimsTetrahydrobiopterin (BH4) is a co-factor of endothelial nitric oxide synthase (eNOS), which is critical to embryonic heart development. We aimed to study the effects of sapropterin (Kuvan(R)), an orally active synthetic form of BH4 on eNOS uncoupling and congenital heart defects (CHDs) induced by pregestational diabetes in mice.\n\nMethodsAdult female mice were induced to pregestational diabetes by streptozotocin and bred with normal males to produce offspring. Pregnant mice were treated with sapropterin or vehicle during gestation. CHDs were identified by histological analysis. Cell proliferation, eNOS dimerization and reactive oxygen species (ROS) production were assessed in the fetal heart.\n\nResultsPregestational diabetes results in a spectrum of CHDs in their offspring. Oral treatment with sapropterin in the diabetic dams significantly decreased the incidence of CHDs from 59% to 27% and major abnormalities, such as atrioventricular septal defect and double outlet right ventricle were absent in the sapropterin treated group. Lineage tracing reveals that pregestational diabetes results in decreased commitment of second heart field progenitors to the outflow tract, endocardial cushions, and ventricular myocardium of the fetal heart. Notably, decreased cell proliferation and cardiac transcription factor expression induced by maternal diabetes were normalized with sapropterin treatment. Furthermore, sapropterin administration in the diabetic dams increased eNOS dimerization and lowered ROS levels in the fetal heart.\n\nConclusionsSapropterin treatment in the diabetic mothers improves eNOS coupling, increases cell proliferation and prevents the development of CHDs in the offspring. Thus, sapropterin may have therapeutic potential in preventing CHDs in pregestational diabetes.

pharmacology and toxicology↗

bioRxiv2018

S-Nitrosylation of STIM1 by neuronal nitric oxide synthase inhibits store-operated Ca2+ entry

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum (ER/SR). However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase (nNOS) is located in the SR of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. nNOS deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione (GSNO) inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49 and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+-depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity, and inhibits SOCE in cardiomyocytes.

physiology↗

bioRxiv2018

Mettl14 is required for mouse post-implantation development by facilitating epiblast maturation

N6-methyladenosine (m6A) is the most prevalent and reversible internal modification of mammalian messenger and noncoding RNAs mediated by specific m6A writer, reader, and eraser proteins. As an m6A writer, the METTL3-METTL14-WTAP complex dynamically regulates m6A modification and plays important roles in diverse biological processes. However, our knowledge about the complete functions of this RNA methyltransferase complex, the contributions of each component to the methylation and their impacts on different biological pathways, are still very limited. Here, by employing both in vivo and in vitro models, we report that METTL14 was indispensable for post-implantation embryonic development by facilitating the conversion from naive to primed state of the epiblast. Depletion of Mettl14 lead to conspicuous embryonic growth retardation from E6.5 mainly as a result of resistance to differentiation, which further lead to embryonic lethality early in gestation. Our data highlight the critical function of METTL14, as an m6A modification regulator, in orchestrating early mouse embryogenesis.

developmental biology↗

bioRxiv2018

Motion Sensing Superpixels (MOSES): A systematic framework to quantify and discover cellular motion phenotypes

Cellular motion is fundamental in tissue development and homeostasis. There is strong interest in identifying factors that affect the interactions of cells in disease but analytical tools for robust and sensitive quantification in varying experimental conditions for large extended timelapse acquisitions is limited. We present Motion Sensing Superpixels (MOSES), a method to systematically capture diverse features of cellular dynamics. We quantify dynamic interactions between epithelial cell sheets using cell lines of the squamous and columnar epithelia in human normal esophagus, Barretts esophagus and esophageal adenocarcinoma and find unique boundary formation between squamous and columnar cells. MOSES also measured subtle changes in the boundary formation caused by external stimuli. The same conclusions of the 190 videos were arrived at unbiasedly with little prior knowledge using a visual motion map generated from unique MOSES motion signatures. MOSES is a versatile framework to measure, characterise and phenotype cellular interactions for high-content screens.

bioinformatics↗

bioRxiv2017

Tumor-specific Causal Inference (TCI): A Bayesian Method for Identifying Causative Genome Alterations within Individual Tumors

Precision medicine for cancer involves identifying and targeting the somatic genome alterations (SGAs) that drive the development of an individual tumor. Much of current efforts at finding driver SGAs have involved identifying the genes that are mutated more frequently than expected among a collection of tumors. When these population-derived driver genes are altered (perhaps in particular ways) in a given tumor, they are posited as driver genes for that tumor. In this technical report, we introduce an alternative approach for identifying causative SGAs, also known as \"drivers\", by inferring causal relationships between SGAs and molecular phenotypes at the individual tumor level. Our tumor-specific causal inference (TCI) algorithm uses a Bayesian method to identify the SGAs in a given tumor that have a high probability of regulating transcriptomic changes observed in that specific tumor. Thus, the method is focused on identifying the tumor specific SGAs that are causing expression changes that are specific to the tumor. Those SGAs that have a high probability of regulating transcriptomic changes related to oncogenic processes are then designated to be the putative drivers of the tumor. In this paper, we describe in detail the TCI algorithm and its implementation.

bioinformatics↗

bioRxiv2017

HTX: a tool for the exploration and visualization of high-throughput image assays

High-throughput screening (HTS) techniques have enabled large scale image-based studies, but extracting biological insights from the imaging data in an exploratory setting remains a challenge. Existing packages for this task either require expert annotations, which can bias the outcome of the study, or are completely unsupervised, failing to leverage the information present in the assay design. We present HTX, an interactive tool to aid in the exploration of large microscopy data sets by allowing the visualization of entire image-based assays according to visual similarities between the samples in an intuitive and navigable manner. Underlying HTX are a collection of novel algorithmic techniques for deep texture descriptor learning, 2D data visualization, adversarial suppression of batch effects, and backprop-based image saliency estimation.\n\nWe demonstrate that HTX can exploit the screen meta-data in order to learn screen-specific image descriptors, which are then used to quantify the visual similarity between samples in the assay. Given these similarities and the different visualization resources of HTX, it is shown that screens of small-molecule libraries on cell data can be easily explored, reproducing the results of previous studies where highly-specific domain knowledge was required.

bioinformatics↗

bioRxiv2017

Genetic load and mutational meltdown in cancer cell populations

ABSRACTLarge and non-recombining genomes are prone to accumulating deleterious mutations faster than natural selection can purge (Mullers ratchet). A possible consequence would then be the extinction of small populations. Relative to most single-cell organisms, cancer cells, with large and non-recombining genomes, could be particularly susceptible to such \"mutational meltdown\". Curiously, deleterious mutations in cancer cells are rarely noticed despite the strong signals in cancer genome sequences. Here, by monitoring single-cell clones from HeLa cell lines, we characterize deleterious mutations that retard cell proliferation. The main mutational events are copy number variations (CNVs), which happen at an extraordinarily high rate of 0.29 events per cell division. The average fitness reduction, estimated to be 18% per mutation, is also very high. HeLa cell populations therefore have very substantial genetic load and, at this level, natural population would likely experience mutational meltdown. We suspect that HeLa cell populations may avoid extinction only after the population size becomes large. Because CNVs are common in most cell lines and cancer tissues, the observations hint at cancer cells vulnerability, which could be exploited by therapeutic strategies.

evolutionary biology↗

bioRxiv2017

Inhibition of mitochondrial fission preserves photoreceptors after retinal detachment

Photoreceptor degeneration is a leading cause of visual impairment worldwide. Separation of neurosensory retina from the underlying retinal pigment epithelium is a prominent feature preceding photoreceptor degeneration in a variety of retinal diseases. Although ophthalmic surgeries have been well developed to restore retinal structures, post-op patients usually experience progressive photoreceptor degeneration and irreversible vision loss that is incurable at present. Previous studies point to a critical role of mitochondria-mediated apoptotic pathway in photoreceptor degeneration, but the upstream triggers remain largely unexplored. In this study, we show that after experimental RD induction, photoreceptors activate dynamin-related protein 1 (Drp1)-dependent mitochondrial fission pathway and subsequent apoptotic cascades. Mechanistically, endogenous ROS is necessary for Drp1 activation in vivo and exogenous ROS insult is sufficient to activate Drp1-dependent mitochondrial fission in cultured photoreceptors. Accordingly, inhibition of Drp1 activity effectively preserves mitochondrial integrity and rescues photoreceptors. Collectively, our data delineates a ROS-Drp1-mitochondria axis that promotes photoreceptor degeneration in retinal diseased models.

neuroscience↗

bioRxiv2017

A Library of Phosphoproteomic and Chromatin Signatures for Characterizing Cellular Responses to Drug Perturbations

Though the added value of proteomic measurements to gene expression profiling has been demonstrated, profiling of gene expression on its own remains the dominant means of understanding cellular responses to perturbation. Direct protein measurements are typically limited due to issues of cost and scale; however, the recent development of high-throughput, targeted sentinel mass spectrometry assays provides an opportunity for proteomics to contribute at a meaningful scale in high-value areas for drug development. To demonstrate the feasibility of a systematic and comprehensive library of perturbational proteomic signatures, we profiled 90 drugs (in triplicate) in six cell lines using two different proteomic assays -- one measuring global changes of epigenetic marks on histone proteins and another measuring a set of peptides reporting on the phosphoproteome -- for a total of more than 3,400 samples. This effort represents a first-of-its-kind resource for proteomics. The majority of tested drugs generated reproducible responses in both phosphosignaling and chromatin states, but we observed differences in the responses that were cell line-and assay-specific. We formalized the process of comparing response signatures within the data using a concept called connectivity, which enabled us to integrate data across cell types and assays. Furthermore, it facilitated incorporation of transcriptional signatures. Consistent connectivity among cell types revealed cellular responses that transcended cell-specific effects, while consistent connectivity among assays revealed unexpected associations between drugs that were confirmed by experimental follow-up. We further demonstrated how the resource could be leveraged against public domain external datasets to recognize therapeutic hypotheses that are consistent with ongoing clinical trials for the treatment of multiple myeloma and acute lymphocytic leukemia (ALL). These data are available for download via the Gene Expression Omnibus (accession GSE101406), and web apps for interacting with this resource are available at https://clue.io/proteomics.\n\nHighlightsO_LIFirst-of-its-kind public resource of proteomic responses to systematically administered perturbagens\nC_LIO_LIDirect proteomic profiling of phosphosignaling and chromatin states in cells for 90 drugs in six different cell lines\nC_LIO_LIExtends Connectivity Map concept to proteomic data for integration with transcriptional data\nC_LIO_LIEnables recognition of unexpected, cell type-specific activities and potential translational therapeutic opportunities\nC_LI

systems biology↗

bioRxiv2017

Mouse hepatitis virus nsp14 exoribonuclease activity is required for resistance to innate immunity

ABSTRACTCoronaviruses (CoV) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that interact with the host innate immune response to facilitate efficient viral replication. CoV non-structural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order Nidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade dsRNA replication intermediates. In this study, we tested the hypothesis that CoV ExoN may also function to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-{beta}) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor deficient (IFNAR-/-) BMMs. ExoN(-) virus replication did not result in IFN-{beta} gene expression, and in the presence of an IFN-{beta}-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to untreated. However, ExoN(-) virus generated from IFN-{beta} pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest MHV ExoN activity is required for resistance to the innate immune response and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.

microbiology↗

bioRxiv2017

Proofreading-deficient coronaviruses adapt over long-term passage for increased fidelity and fitness without reversion of exoribonuclease-inactivating mutations

The coronavirus (CoV) RNA genome is the largest among single-stranded positive sense RNA viruses. CoVs encode a proofreading 3'[->]5'exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in SARS-CoV and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold decreased fidelity, and increased susceptibility to nucleoside analogs. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with WT-MHV. Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication, reduced susceptibility to nucleoside analogs, and increased competitive fitness. However, passage did not select for complete or partial reversion at the ExoN-inactivating mutations. We identified novel amino acid changes within the RNA-dependent RNA polymerase (nsp12-RdRp) and nsp14 of MHV-ExoN(-) P250 that partially account for the observed changes in replication, susceptibility to nucleoside analogs, and competitive fitness observed in the passaged virus population, indicating that additional determinants can compensate for the activities of nsp14-ExoN. Our results suggest that while selection favors restoration of replication fidelity in ExoN(-) CoVs, there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and identify additional candidate proteins that may regulate CoV replication fidelity.\n\nIMPORTANCEUnique among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] are either non-viable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we show that ExoN(-) murine hepatitis virus can adapt over long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogs that is only partially explained by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogs is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

microbiology↗