Biology subjects

Nguyen, A.

Publications and source records attributed to Nguyen, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Serotonergic circuit architecture underlies sex dimorphism in anxiogenic states

The serotonergic system underpins anxiety-like behavior vital for the adaptive response to a new environment. However, our understanding of the neural circuitry underlying the diversity of anxiety-like behaviors across individuals is limited. Whilst previous studies have characterized diverse features related to the neurochemical substrate of serotonergic neurotransmission and behavioral presentation between males and females, no investigations have fully addressed the sexual dimorphism in the structural and functional connectivity of serotonergic circuits. Here, using functional FosTRAP connectivity analysis, we found sex-dependent functional differences in the serotonergic neurons of the dorsal raphe nucleus (DRN), such as enriched connectivity to the amygdala in females, compared to male mice. We also discovered previously unidentified morpho-functional sex-specific differences in DRN serotonergic circuitry that reflects the disparity in behavior presentation. To understand the molecular basis of serotonergic circuit architecture differences between sexes, we leveraged on single-cell RNA sequencing dataset from DRN serotonergic cells taking sex as a biological variable and spotted some gene candidates involved in neural circuit wiring. Altering the expression of the receptor tyrosine kinase Erbb4 in serotonergic circuits, altered the axonal connectivity to downstream targets and shifted sex-specific behavior which. Overall, this study provides an essential foundation to delineate the molecular basis and connectivity pattern mechanisms underlying the serotonergic system-dependent sex-specific behaviors. Since alterations in serotonin function play a vital role in adaptive behavior, as well as various pathologies including chronic anxiety and depression, this study may advance our understanding of sex-biases in presentation and treatment response of neuropsychiatric disorders.

neuroscience↗

bioRxiv2026

Chromatin-Assisted Targeting Enables Precise DNA Methylation Editing in Plants

Precise installation of DNA methylation at selected loci offers a powerful strategy for regulating gene expression without altering DNA sequence, but existing plant epigenome editors are constrained by limited efficiency, locus dependence, and genome-wide off-target methylation. Here, we developed SunTag-MQ1v variants incorporating TRBIP1, which promotes removal of the antagonistic H3K4me3 mark, and CHLAMY, an oligomerizing alpha crystalline domain protein from Chlamydomonas reinhardtii. TRBIP1 enhanced methylation and silencing at the Arabidopsis FWA promoter but caused widespread off-target methylation and severe developmental defects. Adding CHLAMY produced SunTag-CHLAMY-TRBIP1-MQ1v (designated as SunTag-NOVA), which successfully overcame the lethality and widespread off-target effects associated with direct TRBIP1-MQ1v fusions. We demonstrate that CHLAMY drives higher-order oligomerization of the editing complex, which enhances target specificity and mitigates off-target accumulation. SunTag-NOVA robustly installed DNA methylation and repressed transcription at the endogenous FWA, FT and TMM genes with minimal genome-wide off-target consequences. These results show that combining local chromatin modification with controlled effector assembly can improve targeted DNA methylation, and establish SunTag-NOVA as a specific epigenome-editing platform for plants.

plant biology↗

bioRxiv2026

Neutrophil remodeling is associated with human meibomian gland dysfunction and enables IFN-γ- and PAD4-dependent gland obstruction in mice

Meibomian gland dysfunction (MGD), a disorder of the eyelid's modified sebaceous glands, is the leading cause of dry eye disease and ocular surface morbidity, yet the immune mechanisms driving gland obstruction remain poorly defined. In a cross-sectional study of 66 patients with ocular surface inflammation, we used meibography and spectral flow cytometry of tear washes to identify a disease-associated, remodeled neutrophil state whose abundance is associated with gland atrophy. Using single-cell transcriptomics in a murine model of immune-mediated MGD, we revealed a disease-associated neutrophil state that exhibited ocular surface-enrichment, CD14 and ICAM-1 expression, and elevated IFN-{gamma} response and inflammatory signatures. Spatial transcriptomics localized IFN-{gamma} signaling and neutrophil migration signatures to the periglandular compartment. The remodeled neutrophils exhibited PAD4-dependent histone citrullination, with Padi4 deletion reducing NET-associated obstructive plugging, thus identifying PAD4-dependent NETotic activity as their disease-producing output. Inhibition of IFN-{gamma} signaling phenocopied Padi4 deficiency, yet combined disruption of these pathways provided no additive protection, indicating that IFN-{gamma} and PAD4 function as separable required inputs. Remodeled neutrophils accumulated under both conditions, uncoupling disease severity from cell abundance alone. Our findings support immune-mediated obstructive MGD as a mechanistic endotype driven by the IFN-{gamma}- and PAD4-dependent effector output of a remodeled neutrophil state.

immunology↗

bioRxiv2026

Trained Planaria Retain Memories Through Head Regeneration: A Model System for Insights into Non-Neural Memory and Neurodegenerative Diseases

Planaria exhibit remarkable regenerative ability, including the capacity to regrow complete heads and brains after decapitation. Here, we re-investigated whether regenerated planaria can preserve learned avoidance behavior, a phenomenon that has been reported previously but has been difficult to study due to unreliable experimental protocols. Using a light-to-food associative conditioning paradigm, planaria were trained to override their normal photophobic preference and then decapitated. Following a two-week regeneration period, behavioral responses to the conditioned stimulus were re-evaluated. Results indicated that the majority of regenerated planaria retained the learned response, supporting a model in which behavioral patterns can regenerate as well as anatomical patterns. By establishing a consistent, low-cost, and effective protocol for studying memory persistence through regeneration, such work may help inform future research on memory loss, resilience, and recovery in neurodegenerative diseases.

animal behavior and cognition↗

bioRxiv2026

Targeted proteomics of postmortem human brain reveals neurobiologic heterogeneity in Alzheimer's disease using NULISA technology

BackgroundAlzheimers disease (AD) is clinicopathologically heterogeneous. A proportion of patients living with AD present clinically at a younger onset of cognitive symptoms before 65 years old and/or non-amnestic clinical syndromes. Neuropathologically, corticolimbic distribution of neurofibrillary tangle pathology occurs on a continuum with some cases having greater cortical tau pathology relative to limbic regions and others with relatively restricted accumulation in limbic structures. These patterns of corticolimbic tangle distribution are associated with clinical presentation and age at onset. This study sought to examine protein expression differences across the spectrum of clinicopathologic heterogeneity using the NULISA targeted proteomics platform. MethodsA series of thirteen neuropathologically diagnosed AD cases from Mayo Clinic prospectively followed research studies were selected to reflect heterogeneity of clinical syndromes and corticolimbic distribution of tangle pathology. Frozen postmortem brain tissue samples were isolated from inferior parietal cortex and homogenized in RIPA buffer for analysis using Alamar Biosciences NULISA CNS disease 120 panel. Applying a conservative detection threshold of 75% level of detection for the novel application of NULISA in human brain, we evaluated levels of 69 of 129 protein targets across samples. We examined associations between age at onset cognitive symptoms and corticolimbic distribution of tangles (CLix) separately with individual protein targets using linear regression analysis. ResultsAD cases with a younger age at onset had higher measured levels of ubiquitin, while older age was associated with higher levels of total tau, CRH, and NPTX2. Investigations of corticolimbic heterogeneity revealed AD cases with lower CLix score (i.e., cortical predominant distribution of tau) had higher measured p-tau181, p-tau231, ubiquitin, and p62. AD cases with higher CLix (i.e., relative cortical sparing) had higher levels of total tau, CRH, NPTX2, MDH1, and HBA1. Brain-derived total tau consistently showed a stronger association in both models. ConclusionThis work demonstrates the utility of postmortem proteomics for investigating biomarkers associated with AD clinicopathologic heterogeneity. We observed proteomic differences in synapse integrity, tau post-translational modification, and ubiquitination associated with age at symptomatic onset and corticolimbic distribution of tangle pathology.

neuroscience↗

bioRxiv2026

Sustained GnRH Agonism Alters Endocrine Dynamics and Pubertal Progression in Juvenile Rats

PurposeGonadotropin releasing hormone (GnRH) agonists are clinically used to delay pubertal progression by suppressing the hypothalamic-pituitary-gonadal (HPG) axis. While GnRH agonists have long been used clinically, the developmental characterization of HPG axis suppression during puberty remains incompletely understood. Thus, we examined the effects of GnRH receptor agonism in juvenile rats. HypothesisSustained GnRH receptor agonism will result in lower gonadal mass, blunt peripheral pubertal landmarks, and alter hormonal signaling dynamics within the HPG axis. MethodsAnimals received a single injection of extended-release leuprolide acetate depot (LA) or vehicle control on postnatal day (PND) 23. Animals were assessed for body mass and peripheral markers of puberty. On PND 44, animals were euthanized and tissues were evaluated to assess additional markers of pubertal maturation, pituitary gene transcript levels, and hormone concentrations in serum and gonads. ResultsIn females, LA treatment resulted in a smaller gonad size, increased body mass, and less vaginal openings. In males, LA treatment resulted in smaller gonads but did not significantly alter body mass or preputial separation. In the pituitary, LA-treated rats had lower Gnrhr, Fshb, and Lhb transcript levels regardless of sex, while females exhibited higher Cga and Nr5a1. Serum FSH and ACTH were lower in LA-treated animals, and treated females also had lower progestins and androstenedione, and higher LH. ConclusionsLA treatment reduced aspects of pubertal maturation and HPG axis output, with sex specific outcomes. These findings highlight the need for integrated, multi-level approaches to understand how altered GnRH signaling impacts pubertal and long-term physiology.

Developmental Biology↗

bioRxiv2026

Ultramutagenesis through combined Msh2 depletion and proofreading-deficient DNA polymerase ε expression enhances small molecule target identification in forward genetic screens

DNA mismatch repair (MMR) deficiency has been widely utilized in forward genetic screens to identify drug resistance mutations and elucidate the mechanisms of action of cytotoxic small molecules. However, MMR deficiency generates a characteristic mutational signature enriched for C>T transitions that precludes saturation of mutagenesis. We hypothesized that expression of proofreading deficient DNA polymerase epsilon, as has been observed in patients with biallelic MMR deficiency, would enhance mutagenesis and enable identification of new resistance mutations and cytotoxin mechanisms. Here, we combine auxin-inducible degradation of Msh2 with doxycycline-inducible expression of the proofreading-deficient Pol{varepsilon}P286R mutant in murine small cell lung cancer (SCLC) cells. Simultaneous MMR deficiency and mutant Pol{varepsilon} expression markedly increased the emergence of bortezomib-resistant clones relative to either perturbation alone and targeted sequencing of Psmb5 in bortezomib-resistant clones identified recurrent mutations previously reported in chemical mutagenesis-based resistance screens. We next applied this platform to investigate resistance to lurbinectedin, a clinically relevant therapy for SCLC. Whole-exome sequencing revealed recurrent loss-of-function alterations in nucleotide excision repair (NER) genes, most notably Ercc5 and Ercc4, implicating NER deficiency as a major mechanism of lurbinectedin resistance and supporting a central role for NER in mediating lurbinectedin-induced cytotoxicity. Collectively, these findings establish inducible ultramutagenesis as a powerful and versatile platform for the unbiased discovery of drug resistance mechanisms and therapeutic vulnerabilities.

Cancer Biology↗

bioRxiv2026

A conserved hydrophobic interaction governs GPCR-transducer association

A central feature of G protein-coupled receptor (GPCR) desensitization is the direct competition between heterotrimeric G proteins and {beta}-arrestins ({beta}arrs) for an overlapping binding site within the intracellular receptor cavity. Although numerous high-resolution structures of GPCR-transducer complexes exist, the exact nature of this shared site and the molecular basis of transducer competition remain unclear. To investigate this, we employed an interdisciplinary approach integrating systemic mutational mapping, bioinformatics, and structural analysis across multiple classes of GPCRs and their transducers and regulators. We identified two highly conserved leucine residues within both the {beta}arr finger loop and the G C-terminal -helix, which engage a hydrophobic patch on GPCRs formed by TM3, TM5, and TM6 in a nearly identical manner, thereby stabilizing the complexes. Notably, the GPCR kinase N-terminal -helix also contains hydrophobic residues that associate with this same receptor patch and are vital for the GPCR-GRK engagement. Our findings reveal a conserved hydrophobic interface that mediates direct competition among GPCR transducers and regulators suggesting a universal mechanism that governs receptor access and desensitization.

biochemistry↗

bioRxiv2026

Intramolecular loops control SARS-CoV-2 nucleocapsid protein self-association and nucleic acid binding dependent on phosphorylation

The nucleocapsid protein of SARS-CoV-2 scaffolds genomic RNA into ribonucleoprotein complexes (RNP) for assembly in the virion, but also fulfills critical intracellular functions in replication and the suppression of host defense. It is comprised of a folded nucleic acid binding domain (NTD) and a dimerization domain, connected by a disordered linker containing a serine/arginine-rich (SR) region and a leucine-rich sequence (LRS). The switch between intracellular and assembly functions of N-protein is controlled by phosphorylation of the SR region, but the molecular details are unclear. Here we describe a model in which two mutually exclusive intramolecular loops bind the NTD and dynamically control self-association and nucleic acid binding properties dependent on the SR linker phosphorylation state. The model is supported by biophysical properties and interactions of full-length protein, point mutants, and peptide fragments. We find SR linker phosphorylation compacts the protein and inhibits nucleic acid binding and RNP formation, while enhancing self-association through promotion of transient coiled-coils in the LRS of the linker. These changes shift the nucleocapsid protein to a configuration poised for multi-valent interactions that support intracellular functions.

biophysics↗

bioRxiv2025

DINOcell: Learning Generalizable Perturbation Effects through Self-Distillation

AO_SCPLOWBSTRACTC_SCPLOWPredicting cellular responses to therapeutics is a promising approach for novel target discovery. However, state-of-the-art computational models designed to predict perturbation effects struggle to generalize and outperform simple baselines. We present DINOcell, a weakly supervised framework that adapts self-distillation to single-cell transcriptomics for predicting perturbation effects. We demonstrate that DINOcell outperforms baselines in predicting the effects of single gene perturbations. Furthermore, DINOcell accurately predicts non-additive effects of combination perturbations, indicating its capacity to model complex genetic interactions. Finally, we show that DINOcell learns representations that capture biological signals and is a promising, generalizable approach for in silico perturbation modeling, providing a valuable tool for accelerating therapeutic target discovery.

bioinformatics↗

bioRxiv2025

Transcriptional Profiling of D1-MSNs Reveals SIRT1-Dependent and -Independent Responses to Chronic Social Defeat Stress

SIRT1 is a critical regulator of neuronal functions and has been implicated in various neurological disorders, including depression. In a previous study, we demonstrated the pro-depressive roles of SIRT1 in the nucleus accumbens (NAc) using the chronic social defeat stress (CSDS) model, a well-validated preclinical mouse model of depression. We found that SIRT1 modulates synaptic functions and exhibits pro-susceptible effects specifically in D1-medium spiny neurons in the NAc. In this study, we extended our investigation of SIRT1 functions by employing cell-type-specific transcriptomics to compare control and susceptible groups under CSDS. Our results revealed that in wild-type mice with functional SIRT1, cellular metabolism is crucial for inducing susceptibility to depression. Conversely, in the absence of functional SIRT1 expression, circadian clock-controlled transcriptional regulation becomes a key factor. These findings suggest that SIRT1 differentially modulates key pathophysiological mechanisms of depression via its deacetylase functions, highlighting its role in both metabolic and circadian regulation in the brain. This research provides new insights into the molecular underpinnings of depression and identifies potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2025

Integrative epigenomic analysis uncovers asymmetry of enhancer activity in Brassica napus

Non-coding regulatory regions are essential to the determination of gene expression and plant phenotypes. In this work, we investigated the cis-regulatory landscape of a winter type rapeseed, Express617, across multiple sample types. Combining chromatin accessibility, DNA methylation and gene expression, we annotated thousands of novel regulatory elements in the Brassica napus genome. Among those regions, we discovered and functionally characterized super-enhancers, observing an asymmetrical distribution of these regulatory elements favoring the Cn subgenome. Super-enhancer (SE) associated genes were found enriched in tissue identity and responses to stimuli related processes. We further establish and apply an in-silico validation pipeline for super-enhancers, integrating population-level expression analysis and machine learning (ML) models predicting gene expression levels. Almost 50% of the newly identified SE-associated genes had an observed expression higher than the expression levels predicted by the ML model. Moreover, structural variants disrupting super-enhancer elements correlate with a reduction of expression in the associated genes, both consistent with the positive effect of these regulatory regions. These results greatly expand the functional annotation of rapeseed and contribute to a better understanding of the link between regulatory elements and their target genes and processes, providing novel insights and targets for B. napus (epi)-genome editing strategies.

plant biology↗

bioRxiv2025

Adaptive evolution of engineered Saccharomyces cerevisiae in favored and unusual chemical environments

Engineered microbial cells can produce a wide range of industrially relevant chemicals such as pharmaceuticals, fuels, and material precursors. The use of microbial cells for chemical production from renewable resources could replace oil-based chemistry and contribute to tackling global grand challenges of climate warming and resource insufficiency. However, it is underexplored how the chemical production by engineered microbial cells is affected by them being proliferating catalysts exposed to Darwinian selection. All proliferating cells are unavoidably subjected to Darwinian selection which favors fitness beneficial phenotypes that seldom include engineered chemical production. Here, adaptive laboratory evolution was performed to characterize the effect of Darwinian selection on Saccharomyces cerevisiae strains expressing two different heterologous pigment producing pathways, blue-coloured indigoidine and red-coloured bikaverin. S. cerevisiae haploid S288C based strain had the genes for bikaverin synthesis integrated in the same locus as the genes for indigoidine synthesis in haploid and diploid S. cerevisiae CEN.PK-based strains. The two different pigment producing strains were cultivated in rich and synthetic defined (without amino acids) media with respirative galactose as the sole carbon source for [~]200 and [~]175 generations, respectively. While CEN.PK-based lineages rapidly lost indigoidine pigmentation independent of growth medium or ploidy, bikaverin pigmentation in S288C-based lineages was robust. The adaptive solutions detected in S288C-based bikaverin producing lineages involved mutations in the galactose utilization pathway whereas the heterologous indigoidine pathway was recurrently mutated in the corresponding lineages. When the bikaverin producing S288C-based lineages were adaptively evolved on the favored glucose carbon source instead, pigmentation declined. Thus, the robustness of the engineered traits appears dependent on challenges in production environment and availability and fitness benefits of adaptive solutions. Whether or when engineered traits of microbial cells are robust when they proliferate in industrial use has scarcely been assessed. Here light was shed to the factors affecting the adaptive loss of engineered traits to facilitate the development of strains and biotechnological processes, including chemical environments, for robust long-term production.

synthetic biology↗

bioRxiv2025

Sleep deprivation constrains dynamic configurations of integrated and segregated brain states impacting cognitive performance

The breakdown of cognitive control following sleep deprivation is widely recognised, but the physiological mechanisms and brain signatures that produce this vulnerability have not been resolved. Effective cognition relies on large-scale brain networks flexibly reconfiguring between states of integration and segregation. Here we combined functional magnetic resonance imaging (fMRI), electroencephalography (EEG), and electrocardiography (ECG) collected during cognitive tasks under rested wakefulness, after sleep deprivation, and following a recovery nap to test the hypothesis that sleep deprivation constrains this dynamical repertoire and disrupts its physiological regulation. Using time-resolved functional connectivity and graph theory, we show that sleep deprivation increases the distribution of connections across networks, while reducing the temporal variability of between-network connectivity. Furthermore, dynamic fluctuations between integrated and segregated modes of network topology were dampened, with brain regions spending more time in intermediate configurations and showing greater instability of mode transitions. These alterations were tightly linked to behavioural impairment: participants who exhibited greater contraction toward intermediate topologies also showed poorer task accuracy and slower responses. Under well-rested conditions, thalamic activity peaked prior to transitions into integrated states and was suppressed during transitions into segregated states, consistent with a coordinating role in cortical dynamics. Sleep deprivation weakened and delayed this thalamic coupling. Finally, global and regional fMRI fluctuations were elevated after sleep loss, becoming decoupled from cardiac physiology while more strongly coupled to EEG delta power, further linking reduced arousal to constrained network flexibility. Together, these findings show that sleep deprivation narrows the brains dynamical repertoire, due to disrupted thalamic regulation and changes to the physiological integration with cortical networks.

neuroscience↗

bioRxiv2025

A phylogenetic estimate of canine retrotransposition rates based on genome assembly comparisons

BackgroundDue to their history of domestication and breed formation, dogs are a powerful system for studying the phenotypic impact of genetic variation. Comparison of canine genome assemblies show that retrotransposons, mobile elements that mobilize via an RNA intermediate, are a major contributor to canine genetic diversity with an eightfold and 17-fold increase of LINE-1 and SINE differences, respectively, in dogs relative to that found among humans. The frequency of dimorphic retrotransposon insertions among dogs suggests these elements have mobilized at a high rate over recent canine evolution. However, the rate at which new insertions arise has yet to be determined. ResultsWe aligned a collection of genome assemblies derived from four breed dogs, a Dingo, and an American grey wolf to a Greenland grey wolf to identify dimorphic LINE-1 and SINEC insertions. Across our panel of seven canine assemblies, we identified and characterized 7,428 dimorphic LINE-1s and 51,572 dimorphic SINECs. Each assembly differs from the Greenland wolf genome by an average of 3,497 LINE-1s and 25,558 SINECs. Analysis of allele sharing among samples recapitulates known relationships and reveals substantial within-breed variation. Calibrating estimates using a previously estimated single nucleotide mutation rate of 4.5x10-9 per base pair per generation, we estimate that new LINE-1 and SINEC and insertions have occurred at a rate of 1/184 and 1/22 births over recent canine evolution. These estimates are largely consistent across assemblies and breeds. ConclusionsOur phylogenetic estimate of SINEC retrotransposition in canines is approximately twice as large as that estimated for Alu elements in humans, while the canine LINE-1 insertion rate is within the range of human estimates. These data suggest that although SINEC has been an outsized driver of canine genome evolution, the striking levels of canine LINE-1 and SINEC dimorphism mainly reflect high levels of long-standing genetic variation.

genomics↗

bioRxiv2025

Epithelial tumor cells utilize mast cell-derived histamine to regulate perineural invasion

Cancer dissemination by perineural invasion (PNI) is associated with poor outcomes in epithelial malignancies, yet the cellular interactions critical to this process are not fully defined. Single-cell transcriptomic analysis of cutaneous squamous cell carcinoma (cSCC) with PNI highlighted KITLG-KIT signaling activation between keratinocytes and mast cells. Additionally, we observed increased expression of HRH1 on tumor cells, which encodes the H1 histamine receptor. We confirmed these changes in other cancers, including PNI-positive hepatobiliary and oral mucosal tumors. Histamine enhanced tumor invasion into nerve in organoids that recapitulate key features of PNI, an effect rescued by H1-antihistamines. Mechanistically, broad upregulation of matrix metalloproteinases (MMPs) through P38 activation underscored histamine-mediated PNI by enabling collagen degradation in the nerve sheath. Examination of multiple cancer types with PNI showed co-expression of HRH1 and MMP2 on tumor cells as well as type IV collagen loss in involved nerves. Retrospective analysis of >15,000 patient health records in two independent databases demonstrated that H1-antihistamine use improved overall survival and immunotherapy response in cancers that spread by PNI. Together, these data reveal a critical role for mast cells in PNI and present a new therapeutic strategy for targeting this process in epithelial cancers.

cancer biology↗

bioRxiv2025

Social Exclusion Amplifies Behavioral Responses to Physical Pain via Insular Neuromodulation

The "Pain Overlap Theory" (1) proposes that the experience of social pain overlaps with and amplifies the experience of physical pain by sharing parts of the same underlying processing systems (2-6). In humans, the insular cortex has been implicated in this overlap of physical and social pain, but a mechanistic link has not been made (2,4,5,7-9). To determine whether social pain can subsequently impact responses to nociceptive stimuli via convergent electrical signals (spikes) or convergent chemical signals (neuromodulators), we designed a novel Social Exclusion paradigm termed the Fear of Missing Out (FOMO) Task which facilitates a mechanistic investigation in mice. We found that socially-excluded mice display more severe responses to physical pain, disrupted valence encoding, and impaired neural representations of nociceptive stimuli. We performed a systematic biosensor panel and found that endocannabinoid and oxytocin signaling in the insular cortex have opposing responses during trials where mice were attending or not attending to the Social Exclusion events respectively, demonstrating distinct neuromodulatory substrates that underpin different states of Social Exclusion. We also found that intra-insular blockade of oxytocin signaling increased the response to physical pain following Social Exclusion. Together these findings suggest Social Exclusion effectively alters physical pain perception using neuromodulatory signaling in the insular cortex.

neuroscience↗

bioRxiv2025

Evolution of a fuzzy ribonucleoprotein complex in viral assembly

Previously we showed that the genetic diversity of SARS-CoV-2 nucleocapsid (N) protein explores a wide range of biophysical properties facilitated by non-local impact of point mutations to its intrinsically disordered regions (Nguyen et al., 2024). This includes modulation of self-association, such as the creation of a de novo binding interface through the P13L mutation characteristic of Omicron variants. In the present work we focus on the key function of N condensing viral RNA into ribonucleoprotein particles (RNPs) for viral assembly. Lacking high-resolution structural information, biochemical and biophysical approaches have revealed architectural principles of RNPs, which involve cooperative interactions of several protein-protein and protein-RNA interfaces, initiated through oligomerization of conserved transient helices in the central disordered linker of N. Here we study the impact of defining N-protein mutations in variants of concern on RNP formation, using biophysical tools, a virus-like particle assay, and reverse genetics experiments. We find convergent evolution in repeated, independent introduction of amino acid substitutions strengthening existing binding interfaces, compensating for other substitutions that promote viral replication but decrease RNP stability. Furthermore, we show that the P13L mutation of Omicron variants enhances RNP assembly and increases viral fitness. Overall, our data reveal RNP complexes to be highly variable not only in sequence and conformations, but also in thermodynamic and kinetic stability, with its pleomorphism affecting basic architectural principles. We hypothesize that the formation of polydisperse, fuzzy N-RNA clusters with multiple distributed weak binding interfaces optimizes reversible RNA condensation, while supporting host adaptation and allowing for a large sequence space to be explored.

biophysics↗