Biology subjects

Kwon, Y.-C.

Publications and source records attributed to Kwon, Y.-C..

12 recordsLinked to original sources

bioRxiv2026

De novo design of a safe and potent respiratory syncytial virus immuno-focusing antigen

Respiratory syncytial virus (RSV) remains the leading cause of severe respiratory infections in infants, the elderly, and the immunocompromised. Although stabilized full-length pre-fusion (pre-F) protein vaccines are promising, enhanced respiratory disease (ERD) remains a critical safety concern. Here, we used artificial intelligence to design a de novo immuno-focused antigen that structurally preserves the RSV F head region containing critical neutralising epitopes-- site O, II and V-while replacing the non-neutralising stem with a computationally designed scaffold to minimise immunopathological risk. The lead candidate, aRF6, elicited robust protective immunity against RSV in mice and similar immunogenicity in non-human primates without detectable toxicity. Importantly, in stringent ERD-promoting models, aRF6 induced minimal pulmonary pathology and markedly attenuated Th2-biased cytokine responses, outperforming formalin-inactivated RSV and full-length-stabilized pre-F. The results of cryoelectron microscopy confirmed that the aRF6 structure precisely matched the computational predictions. These results demonstrated that computationally designed de novo immuno-focused antigens can yield safe and effective RSV vaccines, thereby providing a rational framework for next-generation vaccine development.

microbiology↗

bioRxiv2026

Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

Long COVID frequently presents with persistent neurological symptoms, including cognitive impairment, fatigue, and sleep disturbances; however, its underlying mechanisms remain unclear. Here, we show that SARS-CoV-2 infection induces lasting cortical neuronal injury and hypothalamic orexin (hypocretin) dysfunction in vivo. In K18-hACE2 and wild-type BALB/c mice, viral RNA persisted in the brain and coincided with focal loss of Neuronal Nuclei (NeuN)-positive cortical neurones beyond acute infection. SARS-CoV-2, but not the influenza A virus, triggered rapid and sustained suppression of hypothalamic orexin expression, defining a virus-specific neuropathological signature. Considering the downregulation of orexin and focal cortical NeuN expression, we showed that augmenting orexin signalling using recombinant orexin-A/B restored NeuN expression in vitro and in vivo. Overall, these findings identify the orexin system as a selective neural vulnerability to SARS-CoV-2 and define orexinergic circuit disruption as a mechanistic axis underlying the neurological manifestations of Long COVID.

microbiology↗

bioRxiv2025

Variation in virion phosphatidylserine content drives differential GAS6 binding among closely related flaviviruses

Many enveloped viruses engage phosphatidylserine (PS) receptors to enter cells, a phenomenon known as "apoptotic mimicry". We previously reported that Zika virus (ZIKV), but not closely-related West Nile virus (WNV) or dengue virus (DENV), utilized AXL to infect cells because only ZIKV could bind the AXL ligand GAS6, a PS-binding protein. In this study, we investigated the mechanisms underlying the differential ability of these viruses to bind GAS6. Although immature virions expose larger patches of the viral membrane than do mature ones, our data show that virion maturity levels did not contribute to GAS6 binding. Surprisingly, while ZIKV contains PS comparable to cellular membranes, PS on WNV and DENV is markedly reduced. These findings explain why only ZIKV can bind GAS6 and provide insights into a novel mechanism by which closely-related flaviviruses differentially utilize cellular entry factors. IMPORTANCEAmong flaviviruses, Zika virus uniquely causes microcephaly and congenital defects. While no flavivirus-specific entry receptors have been identified, they commonly take advantage of phosphatidylserine (PS) receptors to enter cells. Our previous studies revealed that Zika virus uniquely utilizes AXL, found in immune-privileged sites such as the brain and placenta, via binding to its ligand, GAS6. Our current study shows that despite being produced from the same cells, Zika virus has substantially higher PS content than closely related dengue and West Nile viruses, which likely explains Zika viruss unique ability to bind GAS6. These findings provide insight into how closely related flaviviruses can vary substantially in their use of cellular entry factors, potentially contributing to the distinct diseases they cause.

microbiology↗

bioRxiv2025

Liquid-liquid phase separation mediated immune evasion of RSV against OAS-RNase L pathway

Respiratory syncytial virus (RSV) infection is the major cause of severe respiratory illnesses in infants and older adults. RSV forms phase-separated biomolecular condensates called inclusion bodies (IBs), which serve as hubs for viral replication. However, the contribution of IBs to host immune response evasion remains elusive. We report that RSV IBs protect viral RNA from the 2'-5' oligoadenylate synthetase (OAS)-RNase L pathway, a critical antiviral defense mechanism that cleaves viral and cellular RNAs. RSV infection did not activate the OAS-RNase L pathway, and ectopically activated RNase L did not suppress viral replication. In RSV-infected cells, double-stranded RNA (dsRNA) was efficiently sequestered within liquid-liquid phase separation (LLPS)-mediated IBs, rendering its detection challenging. LLPS perturbation caused dsRNA release from IBs into the cytosol. dsRNA extracted from infected cells, which lacked LLPS shielding, triggered OAS-RNase L pathway activation. Thus, LLPS-driven IBs structurally sequester viral RNA, facilitating RSV to evade RNase-dependent genomic RNA degradation mediated by the OAS-RNase L antiviral pathway.

microbiology↗

bioRxiv2024

MEMO: A Micro Memo Sensor Detecting microRNA-RISC using an Accurate Cell-Free Expression Platform

Circulating microRNAs have emerged as potential prognostic biomarkers of various diseases. They serve as micro memos sent out by cells, which scientists can intercept to gain real-time insights into cellular and disease status. However, establishing a standardized microRNA detection platform remains a challenge. Here, we integrate the RNA-induced silencing complex (RISC), which occupies mature microRNAs, into a complex cell-free synthetic genetic circuit using three different RNA polymerases, creating an innovative microRNA biosensor that is sensitive, precise, and cost-effective. The RISC protects the microRNA degradation during isolation and ensures the elimination of false target interactions, enhancing the detection robustness. With a limit of detection of 81 pM, affordability, and the possibility for on-demand use, this system proves to be a strong miRNA sensing tool.

synthetic biology↗

bioRxiv2023

Expanding the Cell-Free Reporter Protein Toolbox by Employing a Split mNeonGreen System to Reduce Protein Synthesis Workload

The cell-free system offers potential advantages in biosensor applications, but their limited substrate supply poses a challenge in balancing enough sensing resources to detect low limits of analyte while providing a robust output signal. In this study, we harnessed split versions of fluorescent proteins, particularly split super-folder green fluorescent protein and mNeonGreen, to improve energy efficiency and enhance detection limit in the cell-free system. A comparative analysis of the expression of 1-10 and eleventh segments of beta strands in both whole-cell and cell-free platforms revealed distinct fluorescence patterns. Moreover, integrating SynZip peptide linkers substantially improved complementation, achieving a fluorescence intensity reaching 73.6% of the full-length protein and a 4.8-fold increase in expression compared to the split system without the SynZip peptide linkers. The split protein reporter system can enable energy-efficient sensing of low analyte levels in the cell-free system, broadening the toolbox of cell-free biosensor repertoire.

synthetic biology↗

bioRxiv2023

Suitability Evaluation of Toehold Switch and EXPAR for Cell-Free MicroRNA Biosensor Development

The development of a robust and cost-effective sensing platform for microRNA (miRNA) is of paramount importance in detecting and monitoring various diseases. Current miRNA detection methods are marred by low accuracy, high cost, and instability. The toehold switch riboregulator has shown promising results in detecting viral RNAs integrated with the cell-free system (CFS). This study aimed to leverage the toehold switch technology to detect miRNA in the CFS and to incorporate the exponential amplification reaction (EXPAR) to bring the detection to clinically relevant levels. We assessed various EXPAR DNA templates under different temperatures and additives to enhance the accuracy of the sensing platform. Furthermore, different structures of toehold switches were tested with either high-concentration synthetic miRNA or EXPAR product to assess sensitivity. Herein, we elucidated the mechanisms of the toehold switch and EXPAR, presented the findings of these optimizations, and discussed the potential benefits and drawbacks of their combined use. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/540462v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1902d64org.highwire.dtl.DTLVardef@94313forg.highwire.dtl.DTLVardef@12d59ddorg.highwire.dtl.DTLVardef@66ecfc_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Zika virus infection induces IL-1β-mediated inflammatory responses by macrophages in the brain of an adult mouse model

During the 2015/16 Zika virus (ZIKV) epidemic, ZIKV associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV-infection mouse model (Ifnar1-/-) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including IL-1{beta}, IL-6, IFN-{gamma}, and TNF-, in the brains of Ifnar1-/- mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days post infection. Furthermore, ZIKV infection induced macrophage infiltration and activation, and augmented IL-1{beta} expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1{beta} secretion. In addition, the expression of complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1{beta}-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also confirmed. Taken together, our results suggest that ZIKV infection of the brain in this animal model augments IL-1{beta} expression in infiltrating macrophages and elicits IL-1{beta}-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. ImportanceZika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection of the mouse brain can induce IL-1{beta}-mediated inflammation and complement activation, contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I IFN receptor IFNAR knockout (Ifnar1-/-) mice owing to the limited mouse model of ZIKV pathogenesis, our conclusion could contribute to understanding ZIKV associated neurological diseases to develop treatment strategies based on these findings for the patients with ZIKV infection.

microbiology↗

bioRxiv2022

Ocular tropism of SARS-CoV-2 with retinal inflammation through neuronal invasion in animal models

Although ocular manifestations are commonly reported in patients with coronavirus disease 2019 (COVID-19), there is currently no consensus on ocular tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To investigate this, we infected K18-hACE2 mice with SARS-CoV-2 using various routes. We observed ocular manifestation and retinal inflammation with cytokine production in the eyes of intranasally (IN) infected mice. An intratracheal (IT) injection resulted in virus spread from the lungs to the brain and eyes via trigeminal and optic nerves. Ocular and neuronal invasion were confirmed by an intracerebral (IC) infection. Notably, eye-dropped (ED) virus did not infect the lungs and was undetectable with time. Using infectious SARS-CoV-2-mCherry clones, we demonstrated the ocular and neurotropic distribution of the virus in vivo by a fluorescence-imaging system. Evidence for the ocular tropic and neuroinvasive characteristics of SARS-CoV-2 was confirmed in wild-type Syrian hamsters. Our data provides further understanding of the viral transmission; SARS-CoV-2 clinical characteristics; and COVID-19 control procedures. SummarySARS-CoV-2 can spread from the respiratory tract to the brain and eyes via trigeminal and optic nerves in animal models. This ocular tropism of SARS-CoV-2 through neuronal invasion likely causes ocular manifestation and retinal inflammation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/488607v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@151c2d5org.highwire.dtl.DTLVardef@ce3aeforg.highwire.dtl.DTLVardef@17f453aorg.highwire.dtl.DTLVardef@99e9c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Characterizing a New Fluorescent Protein for Low Limit of Detection Sensing in the Cell-Free System

Cell-free protein synthesis-based biosensors have been developed as highly accurate, low- cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensors dynamic range must be increased in the system to achieve the low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein - mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder Green Fluorescent Protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce false positives proportion.

synthetic biology↗

bioRxiv2022

SARS-CoV-2 Infection of Microglia Elicits Pro-inflammatory Activation and Apoptotic Cell Death

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in coronavirus disease 2019 (COVID-19) patients. The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we report that SARS-CoV-2 can directly infect human microglia, eliciting M1-like pro-inflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA-seq analysis also revealed that ER stress and immune responses were induced in the early and apoptotic processes in the late phase of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced pro-inflammatory cytokines such as interleukin (IL)-1{beta}, IL-6, and tumour necrosis factor (TNF-), but not the anti-inflammatory cytokine IL-10. After this pro-inflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of pro-inflammatory microglial IL-6 and TNF- and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in COVID-19 patients. IMPORTANCERecent studies reported neurological manifestations and complications in COVID-19 patients, which are associated with neuroinflammation. As microglia are the dominant immune cells in brains, it needs to be elucidate the relationship between neuroinflammation and host immune response of microglia to SARS-CoV-2. Here, we suggest that SARS-CoV-2 can directly infect human microglia with cytopathic effect (CPE) using human microglial clone 3 (HMC3). The infected microglia were promoted to pro-inflammatory activation following apoptotic cell death. This pro-inflammatory activation was accompanied by the high production of pro-inflammatory cytokines, and led to neurotoxic-M1 phenotype polarization. In vivo, murine microglia were infected and produced pro-inflammatory cytokines and provoked a chronic loss using K18-hACE2 mice. Thus, our data present that SARS-CoV-2-infected microglia are potential mediators of neurological problems in COVID-19 patients. In addition, HMC3 cells are susceptible to SARS-CoV-2 and exhibit the CPE, which can be further used to investigate cellular and molecular mechanisms of neuroinflammation reported in COVID-19 patients.

microbiology↗

bioRxiv2021

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alphas small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic. IMPORTANCEThe global pandemic caused by SARS-CoV-2 continues to persist, due in part to mutations that have resulted in the emergence of different variants. Many of these variants have become more virulent and infectious than their ancestral strain, resulting in an ever-increasing spread. However, our virological understanding of these variants remains poor. Here, we directly compared the plaque size, stability, and replication kinetics of four SARS-CoV-2 variants of concern following prolonged incubation at physiological temperatures. Our observations may help to characterize each variant in terms of their interactions with host factors and responses to environmental conditions. We also believe that our evaluations will improve understanding of the emergence of new variants and contribute to controlling their spread.

microbiology↗