Biology subjects

Lee, J.-Y.

Publications and source records attributed to Lee, J.-Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Darapladib, an inhibitor of Lp-PLA2, sensitizes cancer cells to ferroptosis by remodeling lipid metabolism

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis, but how these fatty acids are manipulated in cells is largely unknown. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen identified that darapladib (SB-480848), an inhibitor of Lp-PLA2, synergistically induced ferroptosis with GPX4 inhibitors. Notably, darapladib was able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, Lp-PLA2 was located in the membrane and cytoplasm and suppressed ferroptosis, suggesting the critical role of intracellular Lp-PLA2. Lipidomic analysis showed that phosphatidylethanolamine (PE) species were generally enriched, while lysophosphatidylethanolamine (lysoPE) and free fatty acid levels were reduced, upon darapladib treatment. Finally, combination treatment with darapladib and PACMA31, a GPX4 inhibitor, efficiently inhibited tumor growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

cancer biology↗

bioRxiv2023

Evaluation of antiviral drugs against newly emerged SARS-CoV-2 Omicron variants

The ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activities of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using half maximal inhibitory concentration (IC50) against human isolated 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOC) and compared them with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50 fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9- (BA.2.75.2), 1.2-(B.1.627.2), and 1.4-fold (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, similar to 1.02, 0.88, and 0.67, respectively, of median IC50-fold changes for previous VOC. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. Altogether, these results indicated that the current antivirals retain antiviral efficacy against newly emerged Omicron subvariants, and provide comprehensive information on the antiviral efficacy of these drugs.

microbiology↗

bioRxiv2023

Coupling Flux Balance Analysis with Reactive Transport Modeling through Machine Learning for Rapid and Stable Simulation of Microbial Metabolic Switching

Integration of genome-scale metabolic networks with reactive transport models (RTMs) is an advanced simulation technique that enables predicting the changes of microbial growth and metabolism in space and time. Despite promising demonstrations in the past, computational inefficiency has been pointed out as a critical issue to overcome because it requires repeated implementation of linear programming (LP) to get flux balance analysis (FBA) solutions in every time step and every spatial grid. To address this challenge, we propose a new simulation method where we train/validate artificial neural networks (ANNs) using FBA solutions randomly sampled and incorporate the resulting reduced-order FBA model (represented as algebraic equations) into RTMs as source/sink terms. We demonstrate the efficiency of our method via a case study of Shewanella oneidensis MR-1 strain. During the aerobic growth on lactate, S. oneidensis produces metabolic byproducts (such as pyruvate and acetate), which are subsequently consumed as alternative carbon sources when the preferred ones are depleted. Simulating such intricate dynamics posed a considerable challenge, which we overcame by adopting the cybernetic approach that describes metabolic switches as the outcome of dynamic competition among multiple growth options. In both zero-dimensional batch and one-dimensional column configurations, the ANN-based reduced-order models achieved substantial reduction of computational time by several orders of magnitude compared to the original LP-based FBA models. Importantly, the ANN models produced robust solutions without any special measures to prevent numerical instability. These developments significantly promote our ability to utilize genome-scale networks in complex, multi-physics, and multi-dimensional ecosystem modeling.

systems biology↗

bioRxiv2022

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, ApoE-LPs transport from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and extracellular vesicles (EVs) along the late endosomal trafficking route. Moreover, complexes of ApoE-LPs and CD63-positive EVs were found to be transmitted from cell to cell. Given the important role of ApoE in viral infections, we studied the hepatitis C virus (HCV) and found that the viral replicase protein NS5A is enriched in ApoE-containing intraluminal vesicles. Interaction between NS5A and ApoE is required for the efficient release of EVs containing viral RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders. Author SummaryThe post-Golgi egress pathway of hepatocyte-derived ApoE-containing lipoproteins (ApoE-LPs) is largely unknown. By using integrative imaging analyses, we show that ApoE-LPs are enriched in CD63-positive endosomes suggesting that these endosomes might be a central hub for the storage of ApoE-LPs from which they are released into the circulation. In addition, we provide evidence for the co-egress of ApoE-LPs with extracellular vesicles (EVs) along the late endosomal route and their transfer from cell to cell. This pathway is hijacked by the hepatitis C virus that induces the production of ApoE-associated EVs containing viral RNA. Given the important role of ApoE in multiple metabolic, degenerative and infectious diseases, and the role of EVs in cell-to-cell communication, these results provide important information how perturbations of ApoE might contribute to various pathophysiologies.

microbiology↗

bioRxiv2022

Neuroanatomical substrates in Parkinson's Disease psychosis and their association with serotonergic receptor gene expression: A coordinate-based meta-regression analysis.

BackgroundCommon neural underpinning of Parkinsons Disease (PD) psychosis across different structural magnetic resonance imaging (MRI) studies remains unclear to this day with few studies and even fewer meta-analyses available. ObjectivesOur meta-analysis aimed to identify and summarise studies using MRI approach to identify PD psychosis-specific brain regions and examine the relation between cortical volume loss and dopaminergic and serotonergic receptor density. MethodsPubMed, Web of Science and Embase were searched for MRI studies of PD psychosis (PDP) compared to PD patients without psychosis (PDnP). Seed-based d Mapping with Permutation of Subject Images was applied in the meta-analysis where coordinates were available. Multiple linear regressions to examine the relationship between grey matter volume loss in PDP and receptor gene expression density (extracted from the Allen Human Brain Atlas) were conducted in R. ResultsWe observed lower grey matter volume in parietal-temporo-occipital regions from our meta-analysis (N studies =10, PDP n=211, PDnP, n=298). These results remained significant after adjusting for PD medications and for cognitive scores. Grey matter volume loss in PDP was associated with local expression of 5-HT1a (b=0.109, p=0.012) and 5-HT2a receptors (b=-0.106, p=0.002) also after adjusting for PD medications (5-HT1a, p = 0.005; 5-HT2a, p = 0.001). ConclusionsWidespread cortical volume loss in the parieto-temporo-occipital regions involved in information processing and integration, as well as attention, could result in PD psychosis symptoms. Neurobiological mechanisms implicating serotonergic receptors may also contribute to this condition.

neuroscience↗

bioRxiv2022

Targeting of plasmodesmal proteins requires unconventional signals

Cellular signaling relies on precise spatial localization and dynamic interactions of proteins within the subcellular compartment or niches, including cell-cell contact sites and connections. In plants, both endogenous and pathogenic proteins gained the ability to target plasmodesmata, the membrane-lined cytoplasmic connections, to regulate or exploit cellular signaling across cell wall boundaries. Those include the receptor-like membrane protein PDLP5, a potent regulator of plasmodesmal permeability that generates feed-forward or -back signals vital for plant immunity and root development. However, little is known about what molecular features determine the plasmodesmal association of PDLP5 or other proteins. Notably, although these proteins each have the ability to target plasmodesmata, no protein motifs or sequences have been identified indicative of targeting signals. To address this knowledge gap, we combined machine learning and targeted mutagenesis approaches. Here we report that PDLP5 and its closely related proteins carry novel targeting signals comprising short stretches of amino acid residues. As for PDLP5, it contains two non-redundant, tandemly arranged signals, either of which is sufficient for both localization and biological function regulating viral movement. Strikingly, plasmodesmal targeting signals exhibit little conservation in sequence but are located similarly proximal to the membrane. These novel unconventional features appear to be a common theme in plasmodesmal targeting.

plant biology↗

bioRxiv2022

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation

ObjectiveHepatitis A virus (HAV) infections are considered not to trigger an innate immune response in vivo, in contrast to hepatitis C virus (HCV). This lack of immune induction has been imputed to strong immune counteraction by HAV proteases 3CD and 3ABC. We aimed at elucidating the mechanisms of innate immune induction and counteraction by HAV and HCV in vivo and in vitro. DesignuPA-SCID mice with humanized liver were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by TLR3 and RIG-I/MDA5, respectively. Cleavage of the adaptor proteins TRIF and MAVS was analyzed by transient and stable expression of HAV and HCV proteases and virus infection. ResultsWe detected similar levels of Interferon stimulated genes (ISGs) induction in hepatocytes of HAV and HCV infected human liver chimeric mice. In cell culture, HAV induced ISGs exclusively upon sensing by MDA5 and dependent on LGP2. TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF was not cleaved. ConclusionsHAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates TLR3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV induced antiviral response in hepatocytes. Our results shed new light on induction and counteraction of innate immunity by HAV and HCV and their potential contribution to clearance and persistence. SIGNIFICANCE OF THIS STUDYO_ST_ABSWhat is already known on this topic?C_ST_ABS-- Despite sharing biological and molecular similarities, HAV infections are always cleared while HCV infections persist in most cases. -- In infected chimpanzees HAV does not trigger a strong innate immune response, as opposed to HCV. This has been imputed to the action of HAV proteases abrogating the signalling pathways. -- Physiological in vitro and in vivo models, based on human hepatocytes, to assess HAV and HCV mechanisms of induction and interference of innate immunity are still missing. What this study adds-- HAV induces an innate immune response in vitro and in vivo, in systems with intact signalling pathways and devoid of adaptive immunity. -- HAV 3ABC and 3CD proteases do not abolish the host innate immune response. -- HCV NS3-4A protease disrupts the RLRs pathways, but cannot cleave TRIF and has no impact on TLR3 response. How this study might affect research, practice or policy-- This study offers a comprehensive, side-by-side investigation on HAV and HCV infections in physiological models which recapitulate a cytokine response in the human liver, and allows a precise assessment of the viral interference related to the function of the respective signalling pathways. -- Our results elucidate mechanisms, so far controversial or poorly investigated, thus contributing to our understanding of HAV clearance and HCV persistence.

immunology↗

bioRxiv2022

Combination therapy with nirmatrelvir and molnupiravir improves the survival of SARS-CoV-2 infected mice

As the SARS-CoV-2 pandemic remains uncontrolled owing to the continuous emergence of variants of concern, there is an immediate need to implement the most effective antiviral treatment strategies, especially for risk groups. Here, we evaluated the therapeutic potency of nirmatrelvir, remdesivir, and molnupiravir and their combinations in SARS-CoV-2-infected K18-hACE2 transgenic mice. Systemic treatment of mice with each drug (20 mg/kg) resulted in slightly enhanced antiviral efficacy and yielded an increased life expectancy of only about 20-40% survival. However, combination therapy with nirmatrelvir (20 mg/kg) and molnupiravir (20 mg/kg) in lethally infected mice showed profound inhibition of SARS-CoV-2 replication in both the lung and brain and synergistically improved survival times up to 80% compared to those with nirmatrelvir (P= 0.0001) and molnupiravir (P= 0.0001) administered alone. This combination therapy effectively reduced clinical severity score, virus-induced tissue damage, and viral distribution compared to those in animals treated with these monotherapies. Furthermore, all these assessments associated with this combination were also significantly higher than that of mice receiving remdesivir monotherapy (P= 0.0001) and the nirmatrelvir (20 mg/kg) and remdesivir (20 mg/kg) combination (P= 0.0001), underscored the clinical significance of this combination. By contrast, the nirmatrelvir and remdesivir combination showed less antiviral efficacy, with lower survival compared to nirmatrelvir monotherapy, demonstrating the inefficient therapeutic effect of this combination. The combination therapy with nirmatrelvir and molnupiravir contributes to alleviated morbidity and mortality, which can serve as a basis for the design of clinical studies of this combination in the treatment of COVID-19 patients. IMPORTANCESince SARS-CoV-2 spread rapidly with the emergence of new variants of concerns, it is necessary to develop effective treatment strategies to treat elderly individuals and those with comorbidities. Antiviral therapy using a combination of drugs is more effective in eradicating viruses and will undoubtedly improve the clinical outcome and survival probability of hospitalized SARS-CoV-2 patients. In the current study, we observed three FDA-approved antivirals nirmatrelvir, remdesivir, and molnupiravir have therapeutic significance with moderate survival for their monotherapies against SARS-CoV-2 infected K18-hACE2 mouse model. The combination of nirmatrelvir and molnupiravir showed significant antiviral activity and a higher survival rate of approximately 80%, providing in vivo evidence of the potential utility of this combination. In contrast, nirmatrelvir and remdesivir combination showed less antiviral potency and emphasized the ineffective significance with less survival. The current study suggests that the nirmatrelvir and molnupiravir combination is an effective drug regimen strategy in treating SARS-CoV-2 patients.

microbiology↗

bioRxiv2022

Structural analysis of cholesterol binding and sterol selectivity by ABCG5/G8

The ATP-binding cassette (ABC) sterol transporters are responsible for maintaining cholesterol homeostasis in mammals by participating in reverse cholesterol transport (RCT) or transintestinal cholesterol efflux (TICE). The heterodimeric ABCG5/G8 carries out selective sterol excretion, preventing the abnormal accumulation of plant sterols in human bodies, while homodimeric ABCG1 contributes to the biogenesis and metabolism of high-density lipoproteins. A sterol-binding site on ABCG5/G8 was proposed at the interface of the transmembrane domain and the core of lipid bilayers. In this study, we have determined the crystal structure of ABCG5/G8 in a cholesterol-bound state. The structure combined with amino acid sequence analysis shows that in the proximity of the sterol-binding site, a highly conserved phenylalanine array supports functional implications for ABCG cholesterol/sterol transporters. Lastly, in silico docking analysis of cholesterol and stigmasterol (a plant sterol) suggests sterol-binding selectivity on ABCG5/G8, but not ABCG1. Together, our results provide a structural basis for cholesterol binding on ABCG5/G8 and the sterol selectivity by ABCG transporters.

biochemistry↗

bioRxiv2022

Disease diagnostics using machine learning of immune receptors

Clinical diagnosis typically incorporates physical examination, patient history, and various laboratory tests and imaging studies, but makes limited use of the human systems own record of antigen exposures encoded by receptors on B cells and T cells. We analyzed immune receptor datasets from 593 individuals to develop MAchine Learning for Immunological Diagnosis (Mal-ID), an interpretive framework to screen for multiple illnesses simultaneously or precisely test for one condition. This approach detects specific infections, autoimmune disorders, vaccine responses, and disease severity differences. Human-interpretable features of the model recapitulate known immune responses to SARS-CoV-2, Influenza, and HIV, highlight antigen-specific receptors, and reveal distinct characteristics of Systemic Lupus Erythematosus and Type-1 Diabetes autoreactivity. This analysis framework has broad potential for scientific and clinical interpretation of human immune responses.

immunology↗

bioRxiv2022

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells

With the emergence of multiple highly transmissible SARS-CoV-2 variants during the recent pandemic, the comparison of their infectivity has become a substantially critical issue for public health. However, a direct assessment of these viral characteristics has been challenging due to the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing techniques to facilitate the evaluation. In a proof-of-concept study using the assay with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our results demonstrate that the Omicron (BA.1) variant is 3-5-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the early phase of infection. To our knowledge, this study provides the first direct measurement of the infectivity of the Omicron variant and new experimental procedures that can be applied for monitoring newly emerging viral variants.

microbiology↗

bioRxiv2022

A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes, that do not include the highly prevalent gt1b, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, but cell-to-cell spreading efficiency was not higher as in other isolates like JFH-1. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is the first efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Author summaryChronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibly to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating the first efficient full-replication cycle cell culture model for highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is not only an important novel development facilitating future efforts in vaccine development. It also provides novel perspectives towards our understanding how liver transplantation drives the evolution of viral isolates with high replication capacity, which might contribute to direct pathogenesis of HCV infection.

microbiology↗

bioRxiv2022

A global lipid map reveals host dependency factors conserved across SARS-CoV-2 variants

A comprehensive understanding of host dependency factors for SARS-CoV-2 remains elusive. We mapped alterations in host lipids following SARS-CoV-2 infection using nontargeted lipidomics. We found that SARS-CoV-2 rewires host lipid metabolism, altering 409 lipid species up to 64-fold relative to controls. We correlated these changes with viral protein activity by transfecting human cells with each viral protein and performing lipidomics. We found that lipid droplet plasticity is a key feature of infection and that viral propagation can be blocked by small-molecule glycerolipid biosynthesis inhibitors. We found that this inhibition was effective against the main variants of concern (alpha, beta, gamma, and delta), indicating that glycerolipid biosynthesis is a conserved host dependency factor that supports this evolving virus.

microbiology↗

bioRxiv2022

Metabolomic, photoprotective, and photosynthetic acclimatory responses to post-flowering drought in sorghum

Climate change is globally affecting rainfall patterns, necessitating the improvement of drought tolerance in crops. Sorghum bicolor is a drought-tolerant cereal capable of producing high yields under water scarcity conditions. Functional stay-green sorghum genotypes can maintain green leaf area and efficient grain filling in terminal post-flowering water deprivation, a period of ~10 weeks. To obtain molecular insights into these characteristics, two drought-tolerant genotypes, BTx642 and RTx430, were grown in control and terminal post-flowering drought field plots in the Central Valley of California. Photosynthetic, photoprotective, water dynamics, and biomass traits were quantified and correlated with metabolomic data collected from leaves, stems, and roots at multiple timepoints during drought. Physiological and metabolomic data was then compared to longitudinal RNA sequencing data collected from these two genotypes. The metabolic response to drought highlights the uniqueness of the post-flowering drought acclimation relative to pre-flowering drought. The functional stay-green genotype BTx642 specifically induced photoprotective responses in post-flowering drought supporting a putative role for photoprotection in the molecular basis of the functional stay-green trait. Specific genes are highlighted that may contribute to post-flowering drought tolerance and that can be targeted in crops to maximize yields under limited water input conditions. HighlightPathways contributing to the long-term maintenance of photosynthetic activity in terminal post-flowering drought are revealed by a comprehensive approach combining in-field photosynthetic physiological analysis, metabolomics, and transcriptomics.

plant 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

The in vitro and in vivo potency of CT-P59 against Delta and its associated variants of SARS-CoV-2

The Delta variant originally from India is rapidly spreading across the world and causes to resurge infections of SARS-CoV-2. We previously reported that CT-P59 presented its in vivo potency against Beta and Gamma variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on the Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal study. CT-P59 showed reduced antiviral activity but enabled neutralization against Delta, Epsilon, and Kappa variants in cells. In line with in vitro results, the mouse challenge experiment with the Delta variant substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against the Delta variant infection, hinting that CT-P59 has therapeutic potency for patients infected with Delta and its associated variants. HighlightsO_LICT-P59 exerts the antiviral effect on authentic Delta, Epsilon and Kappa variants in cell-based experiments. C_LIO_LICT-P59 showed neutralizing potency against variants including Delta, Epsilon, Kappa, L452R, T478K and P681H pseudovirus variants. C_LIO_LIThe administration of clinically relevant dose of CT-P59 showed in vivo C_LIO_LIprotection against Delta variants in animal challenge experiment. C_LI

microbiology↗

bioRxiv2021

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2020

RORα enforces stability of the T-helper-17 cell effector program

T helper 17 (Th17) cells regulate mucosal barrier defenses, but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been described, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor ROR{gamma}t is critical for Th17 cell differentiation, but a distinct role of the closely-related ROR, which is co-expressed in Th17 cells, is not known. Here we demonstrate that, although dispensable for Th17 cell differentiation, ROR governs optimal Th17 responses in peripheral tissues. Thus, the absence of ROR in T cells led to significant reductions in both ROR{gamma}t expression and effector function amongst Th17 cells, due to need for cooperative ROR and ROR{gamma}t binding to a newly-identified Rorc enhancer element that is essential for Th17 lineage maintenance in vivo. Altogether, these data point to a non-redundant role of ROR in Th17 lineage maintenance via reinforcement of the ROR{gamma}t transcriptional program.

immunology↗