Intracellular RNA Research Articles

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Toward understanding lipid reorganization in RNA lipid nanoparticles in acidic environments

The use of lipid nanoparticles (LNPs) for therapeutic RNA delivery has gained significant interest, particularly highlighted by recent milestones such as the approval of Onpattro and two mRNA-based SARS-CoV-2 vaccines. However, despite substantial advancements in this field, our understanding of the structure and internal organization of RNA-LNPs -and their relationship to efficacy, both in vitro and in vivo- remains limited. In this study, we present a coarse-grained molecular dynamics (MD) approach that allows for the simulations of full-size LNPs. By analyzing MD-derived structural characteristics in conjunction with cellular experiments, we investigate the effect of critical parameters, such as pH and composition, on LNP structure and potency. Additionally, we examine the mobility and chemical environment within LNPs at a molecular level. Our findings highlight the significant impact that LNP composition and internal molecular mobility can have on key stages of LNP-based intracellular RNA delivery.

Lambda carrageenan displays antiviral activity against the infectious pancreatic necrosis virus (IPNV) by inhibiting viral replication and enhancing innate immunity in salmonid cells

Sulfated polysaccharide lambda carrageenan (λ-CGN) was evaluated for its antiviral effect against IPNV using the in vitro infection model of CHSE-214 salmonid cells. A plaque reduction lysis assay revealed that λ-CGN has an IC50 of 0.9 μg⋅mL−1, CC50 > 128 μg⋅mL−1 and a Selectivity Index (SI) > 142. In comparison, iota, kappa carrageenans and lambda oligo-carrageenan (λ-OC) were less effective than λ-CGN against IPNV. λ-CGN showed no virucidal activity when applied directly to viral particles. Time of addition experiments showed that pre-treatment, co-treatment, and post-treatment with λ-CGN significantly reduced viral RNA copies in the cell supernatant. Additionally, a decrease in intracellular viral RNA was observed with pre-treatment and post-treatment, indicating an impact on different stages of viral replication. Polyacrylamide gel electrophoresis of IPNV genomic RNA in presence of λ-CGN showed a reduction in the level of viral genomic RNA. Confocal microscopy confirmed the intracellular localization of λ-CGN, suggesting that λ-CGN may inhibit the synthesis of IPNV genomic RNA. Moreover, cells pre-treated with λ-CGN showed an increased expression of innate immunity genes CXCL11, IL1β, IFNa, and IRF3. These findings highlight the need for further research to confirm the in vivo pharmacological potential of λ-CGN as a new antiviral agent in salmonids.

Diverse functions of DEAD-box proteins in oligodendrocyte development, differentiation, and homeostasis.

Oligodendrocytes, a type of glial cell in the central nervous system, have a critical role in the formation of myelin around axons, facilitating saltatory conduction, and maintaining the integrity of nerve axons. The dysregulation of oligodendrocyte differentiation and homeostasis have been implicated in a wide range of neurological diseases, including dysmyelinating disorders (e.g., Pelizaeus-Merzbacher disease), demyelinating diseases (e.g., multiple sclerosis), Alzheimer's disease, and psychiatric disorders. Therefore, unraveling the mechanisms of oligodendrocyte development, differentiation, and homeostasis is essential for understanding the pathogenesis of these diseases and the development of therapeutic interventions. Numerous studies have identified and analyzed the functions of transcription factors, RNA metabolic factors, translation control factors, and intracellular and extracellular signals involved in the series of processes from oligodendrocyte fate determination to terminal differentiation. DEAD-box proteins, multifunctional RNA helicases that regulate various intracellular processes, including transcription, RNA processing, and translation, are increasingly recognized for their diverse roles in various aspects of oligodendrocyte development, differentiation, and maintenance of homeostasis. This review introduces the latest insights into the regulatory networks of oligodendrocyte biology mediated by DEAD-box proteins.

Hyperfunctional T cell responses unchecked by regulatory T cells are unable to resolve hepaciviral infection without humoral contribution

The most recent T cell-based vaccine against HCV tested in humans failed to swing the pendulum from chronicity to resolution, despite eliciting cellular responses in most individuals. These results naturally evoke the question of whether hyperactivated responses of a single adaptive immune arm are capable of inducing HCV clearance or if coordinated efforts between antibodies and T cells are indeed necessary. Here, we sought to address this point in determining whether the suppression of antiviral T cell and IgG responses by regulatory T cells (Tregs) is a critical prerequisite of delayed viral clearance or overt chronicity. Using a surrogate model of HCV infection, rodent hepacivirus (RHV) infection in mice, we utilized Foxp3-DTR mice to assess how Tregs modulate the generation of acute antiviral adaptive immune responses and indirectly dictate infection fate via intracellular flow cytometry staining, ELISA, RNA sequencing, and qPCR. Transient depletion of Tregs prior to infection decreased viral-specific CD4+ T cell function, IgG production, and delayed viral clearance. In contrast, transient Treg depletion after infection increased both T cell functionality and IgG production, thereby facilitating accelerated viral clearance. Hyperactivated T cells, achieved via transient Treg depletion, were unable to clear the virus as an isolated effector arm without the help of viral-specific IgG production. Tregs control the outcome of RHV infection via direct modulation of CD4+ T cells and IgG production. Hyperactivated T cell responses are incapable of compensating for experimentally induced lack of antibodies, further reinforcing the notion of cooperative interplay between adaptive immune arms in facilitating hepaciviral clearance. Herein, we demonstrate how timing of regulatory T cell depletion determines the fate of effector T cells, humoral responses, and the kinetics of viral clearance. Our observations provide direct evidence that functional T cell responses are incapable of compensating for suboptimal humoral responses to facilitate viral resolution. Our results imply that future HCV vaccine regimens should not solely rely on eliciting focused responses of a single effector arm, but rather incorporate immunogens capable of inducing durable features of both humoral and cellular memory.

RNA infrastructure profiling illuminates transcriptome structure in crowded spaces.

RNAs fold into compact structures and undergo protein interactions in cells. These occluded environments can block reagents that probe the underlying RNAs. Probes that can analyze structure in crowded settings can shed light on RNA biology. Here, we employ 2'-OH-reactive probes that are small enough to access folded RNA structure underlying close molecular contacts within cells, providing considerably broader coverage for intracellular RNA structural analysis. The data are analyzed first with well-characterized human ribosomal RNAs and then applied transcriptome-wide to polyadenylated transcripts. The smallest probe acetylimidazole (AcIm) yields 80% greater structural coverage than larger conventional reagent NAIN3, providing enhanced structural information in hundreds of transcripts. The acetyl probe also provides superior signals for identifying m6A modification sites in transcripts, particularly in sites that are inaccessible to a standard probe. Our strategy enables profiling RNA infrastructure, enhancing analysis of transcriptome structure, modification, and intracellular interactions, especially in spatially crowded settings.

A new approach for extracellular RNA recovery from Rhodovulum sulfidophilum

The development of RNA-based drugs is highly pursued due to the possibility of creating viable and effective therapies. However, their translation to clinical practice strongly depends on efficient technologies to produce substantial levels of these biomolecules, with high purity and high quality. RNAs are commonly produced by chemical or enzymatic methods, displaying these limitations. In this sense, recombinant production arises as a promising, cost-effective method, allowing large-scale production of RNA. Rhodovulum sulfidophilum (R. sulfidophilum), a marine purple bacterium, offers the advantage of RNA secretion into the extracellular medium, which contains low levels of RNases and other impurities. Therefore, RNA recovery can be simplified compared to standard extraction protocols involving cell lysis, resulting in a more clarified sample and an improved downstream process. In this work, R. sulfidophilum was transformed with a plasmid DNA encoding pre-miR-29b-1, which is known to be involved in the Alzheimer's disease pathway. After production, the pre-miR-29b-1 was recovered through different extraction methods to verify the most advantageous one. A protocol for extracellular RNA recovery was then established, revealing to be a simpler and less time-consuming method, reducing around 16 h in execution time and the quantity of reagents needed when compared to other established methods. The new strategy brings the additional advantage of eliminating the toxic organic compounds routinely used in intracellular RNA extractions. Overall, the optimized process described here, using isopropanol as the precipitation agent, offers a greener, safer, and faster alternative for recombinant RNA recovery and concentration, with an extracellular RNA recovery of 7 μg/mL and target pre-miRNA-29b-1 recovery of 0.7 μg/L of medium.

DNase II Can Efficiently Digest RNA and Needs to Be Redefined as a Nuclease.

DNase II, identified in 1947 and named in 1953, is an acidic DNA endonuclease prevalent across organisms and crucial for normal growth. Despite its expression in nearly all human tissues, as well as its biological significance, DNase II's detailed functions and corresponding mechanisms remain unclear. Although many groups are trying to figure this out, progress is very limited. It is very hard to connect its indispensability with its DNA cleavage activity. In this study, we find that DNase II secreted to saliva can digest RNA in mildly acidic conditions, prompting us to hypothesize that salivary DNase II might digest RNA in the stomach. This finding is consistent with the interesting discovery reported in 1964 that RNA could inhibit DNase II's activity, which has been largely overlooked. This RNA digestion activity is further confirmed by using purified DNase II, showing activity to digest both DNA and RNA effectively. Here, we suggest redesignating DNase II as DNase II (RNase). The biological functions of DNase II are suggested to recycle intracellular RNA or digest external nucleic acids (both RNA and DNA) as nutrients. This discovery may untangle the mystery of DNase II and its significant biofunctions.

M7GRegpred: substrate prediction of N7-methylguanosine (m7G) writers and readers based on sequencing features.

N7-Methylguanosine (m7G) is important RNA modification at internal and the cap structure of five terminal end of message RNA. It is essential for RNA stability of RNA, the efficiency of translation, and various intracellular RNA processing pathways. Given the significance of the m7G modification, numerous studies have been conducted to predict m7G sites. To further elucidate the regulatory mechanisms surrounding m7G, we introduce a novel bioinformatics framework, m7GRegpred, designed to forecast the targets of the m7G methyltransferases METTL1 and WDR4, and m7G readers QKI5, QKI6, and QKI7 for the first time. We integrated different features to build predictors, with AUROC scores of 0.856, 0.857, 0.780, 0.776, 0.818 for METTL1, WDR4, QKI5, QKI6, and QKI7, respectively. In addition, the effect of window lengths and algorism were systemically evaluated in this work. The finial model was summarized in a user-friendly webserver: http://modinfor.com/m7GRegpred/. Our research indicates that the substrates of m7G regulators can be identified and may potentially advance the study of m7G regulators under unique conditions.

Anti-SARS-CoV-2 gapmer antisense oligonucleotides targeting the main protease region of viral RNA

Given the worldwide risk for the outbreak of emerging/re-emerging respiratory viruses, establishment of new antiviral strategies is greatly demanded. In this study, we present a scheme to identify gapmer antisense oligonucleotides (ASOs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA that efficiently inhibit viral replication. We synthesized approximately 300 gapmer ASOs designed to target various SARS-CoV-2 RNA regions and evaluated their activity in cell-based assays. Through a multistep screening in cell culture systems, we identified that ASO#41, targeting the coding region for viral main protease, reduced SARS-CoV-2 RNA levels in infected cells and inhibited virus-induced cytopathic effects. Antiviral effect of ASO#41 was also observed in iPS cell-derived human lung organoids. ASO#41 depleted intracellular viral RNAs during genome replication in an endogenous RNaseH-dependent manner. ASO#41 showed a wide range of antiviral activity against SARS-CoV-2 variants of concern including Alpha, Delta, and Omicron. Intranasal administration to mice exhibited intracellular accumulation of ASO#41 in the lung and significantly reduced the viral infectious titer, with milder body weight loss due to SARS-CoV-2 infection. Further chemical modification with phosphoryl guanidine-containing backbone linkages provided an elevation of anti-SARS-CoV-2 activity, with 23.4 nM of 50% antiviral inhibitory concentration, one of the strongest anti-SARS-CoV-2 ASOs reported so far. Our study presents an approach to identify active ASOs against SARS-CoV-2, which is potentially useful for establishing an antiviral strategy by targeting genome RNA of respiratory viruses.

Interferon-stimulated gene (ISG12a) suppresses hepatitis B virus replication in Huh 7 cells line

Hepatitis B virus (HBV) and its associated chronic liver disease pose a significant health hazard. Chronic HBV infection is a major contributor to the development of liver cirrhosis, liver fibrosis, and liver cancer. This study focused on ISG12a and its inhibitory effect in Huh7 cells on HBV gene expression and replication. The mammalian hepatoma cells Huh7 were transfected with the pHBV1.3 vector (pCAGGS) or HA-tagged ISG12a to determine the overexpression, to test this hypothesis further, we did knockdown or silencing by transducing Huh7 cells with lentiviruses containing shRNAs targeting ISG12a or scramble control shRNA (shControl). The expression of ISG12a was evaluated through western blot analysis. The HBsAg and HBeAg secretion in the Huh7 cells’ culture media was examined using an ELISA test. The qRT-PCR confirmed the mRNA and 3.5 mRNA Kb of ISG12a. The HBV total RNA was extracted and evaluated through a Northern blot. The isolated DNA was detected through qPCR. Additionally, a mechanistic study of enhancer II (EnhII/Cp) was examined through luciferase reporter testing. Our research findings indicate that ISG12a, an interferon-stimulated gene (ISG), plays a crucial role in suppressing the replication and gene expression of HBV. According to our study using the Huh7 cell system, overexpression of ISG12a resulted in a notable reduction in HBV protein levels as well as intracellular core-associated DNA and RNA levels. On the other hand, silencing ISG12a in Huh7 cells resulted in increased HBV RNA transcripts, DNA, and secreted proteins, indicating that ISG12a plays a role in suppressing HBV replication. Additionally, the research revealed that ISG12a inhibits the function of the EnhII/Cp promoter, which results in reduced HBV gene expression. The EnhII/Cp promoter is involved in regulating HBV gene expression, and the study showed that ISG12a restricts its activity. ISG12a may have a regulatory role in controlling the expression of HBV genes. These findings highlight the importance of ISG12a in HBV gene expression and provide valuable insights for understanding its antiviral function.

Robust CXCL10/IP-10 and CCL5/RANTES Production Induced by Tick-Borne Encephalitis Virus in Human Brain Pericytes Despite Weak Infection.

Tick-borne encephalitis virus (TBEV) targets the central nervous system (CNS), leading to potentially severe neurological complications. The neurovascular unit plays a fundamental role in the CNS and in the neuroinvasion of TBEV. However, the role of human brain pericytes, a key component of the neurovascular unit, during TBEV infection has not yet been elucidated. In this study, TBEV infection of the primary human brain perivascular pericytes was investigated with highly virulent Hypr strain and mildly virulent Neudoerfl strain. We used Luminex assay to measure cytokines/chemokines and growth factors. Both viral strains showed comparable replication kinetics, peaking at 3 days post infection (dpi). Intracellular viral RNA copies peaked at 6 dpi for Hypr and 3 dpi for Neudoerfl cultures. According to immunofluorescence staining, only small proportion of pericytes were infected (3% for Hypr and 2% for Neudoerfl), and no cytopathic effect was observed in the infected cells. In cell culture supernatants, IL-6 production was detected at 3 dpi, together with slight increases in IL-15 and IL-4, but IP-10, RANTES and MCP-1 were the main chemokines released after TBEV infection. These chemokines play key roles in both immune defense and immunopathology during TBE. This study suggests that pericytes are an important source of these signaling molecules during TBEV infection in the brain.

Correlative Super-resolution Optical and Electron Microscopic Imaging of Intracellular Ribosomal RNA by a Terpyridine Iridium(III) Complex.

Ribosomal RNA (rRNA) plays a vital role in binding amino acids together, which dictates the primary structure of a protein. Visualization of its intracellular distribution and dynamics during protein synthesis enables a better understanding of the correlated biological essence. However, appropriate tools targeting live cell rRNA that are capable of multimodal imaging at the nanoscale are still lacking. Here, we rationally designed a series of terpyridine ammonium iridium(III) complexes, one of which is capable of selectively labeling rRNA in living cells. Its metal core and photostable nature allow further super-resolution STED imaging of rRNA found on the rough endoplasmic reticulum at a ∼40 nm resolution that is well correlated under correlative light and electron microscopy (CLEM). Interestingly, the Ir(III) complex demonstrated rRNA dynamics in living cells while boosting protein synthesis at the nanoscale. Our work offers a versatile tool to visualize rRNA synchronously under optical and electron microscopy, which provides a better understanding of rRNA evolution in living systems.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wild-type human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.

CD72 is an inhibitory pattern recognition receptor that recognizes ribosomes and suppresses production of anti-ribosome autoantibody

B cell responses to nucleic acid-containing self-antigens that involve intracellular nucleic acid sensors play a crucial role in autoantibody production in SLE. CD72 is an inhibitory B cell co-receptor that down-regulates BCR signaling, and prevents the development of SLE. We previously showed that CD72 recognizes the RNA-containing self-antigen Sm/RNP, a target of SLE-specific autoantibodies, and induces B cell tolerance to Sm/RNP by specifically inhibiting B cell response to this self-antigen. Here, we address whether CD72 inhibits B cell response to ribosomes because the ribosome is an RNA-containing self-antigen and is a target of SLE-specific autoantibodies as well as Sm/RNP. We demonstrate that CD72 recognizes ribosomes as a ligand, and specifically inhibits BCR signaling induced by ribosomes. Although conventional protein antigens by themselves do not induce proliferation of specific B cells, ribosomes induce proliferation of B cells reactive to ribosomes in a manner dependent on RNA. This proliferative response is down-regulated by CD72. These results suggest that ribosomes activate B cells by inducing dual signaling through BCR and intracellular RNA sensors and that CD72 inhibits B cell response to ribosomes. Moreover, CD72−/− but not CD72+/+ mice spontaneously produce anti-ribosome autoantibodies. Taken together, CD72 induces B cell self-tolerance to ribosomes by recognizing ribosomes and inhibiting RNA-dependent B cell response to this self-antigen. CD72 appears to prevent development of SLE by inhibiting autoimmune B cell responses to multiple RNA-containing self-antigens. Because these self-antigens but not protein self-antigens induce RNA-dependent B cell activation, self-tolerance to RNA-containing self-antigens may require a distinct tolerance mechanism mediated by CD72.

Curcumin reduces myocardial ischemia-reperfusion injury, by increasing endogenous H2S levels and further modulating m6A.

Our previous research shows that Curcumin (CUR) attenuates myocardial ischemia-reperfusion injury (MIRI) by reducing intracellular total RNA m6A levels. However, the mechanism remains unknown. For ischemia-reperfusion (IR), H9c2 cells were cultured for 6h in serum-free low-glycemic (1g/L) medium and a gas environment without oxygen, and then cultured for 6h in high-glycemic (4.5g/L) medium supplemented with 10% FBS and a 21% oxygen environment. The effects of different concentrations of CUR (5, 10, and 20 µM) treatments on signaling molecules in conventionally cultured and IR-treated H9c2 cells were examined. CUR treatment significantly up-regulated the H2S levels, and the mRNA and protein expression of cystathionine γ-lyase (CSE), and down-regulated the mRNAs and proteins levels of thiosulfate sulfurtransferase (TST) and ethylmalonic encephalopathy 1 (ETHE1) in H9c2 cells conventionally cultured and subjected to IR. Exogenous H2S supply (NaHS and GYY4137) significantly reduced intracellular total RNA m6A levels, and the expression of RNA m6A "writers" METTL3 and METTL14, and increased the expression of RNA m6A "eraser" FTO in H9c2 cells conventionally cultured and subjected to IR. CSE knockdown counteracted the inhibitory effect of CUR treatment on ROS production, promotion on cell viability, and inhibition on apoptosis of H9c2 cells subjected to IR. CUR attenuates MIRI by regulating the expression of H2S level-regulating enzymes and increasing the endogenous H2S levels. Increased H2S levels could regulate the m6A-related proteins expression and intracellular total RNA m6A levels.

Inhibitory effect of small-molecule compound AM679 targeting elongation-factor binding protein 2 on hepatitis B virus in vitro

Objective: To explore the antiviral activity of the small-molecule compound AM679 in hepatitis B virus (HBV) replication and infection cell models. Methods: The positive regulatory effect of AM679 on EFTUD2 expression was validated by qPCR and Western blotting. HepAD38 and HepG2-NTCP cells were treated with AM679 (0.5, 1, and 2 nmol/L). Negative control, positive control, and AM679 combined with the entecavir group were set up. HBV DNA intra-and extracellularly, as well as the expression levels of intracellular HBV total RNAs and 3.5kb-RNA changes, were detected with qPCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels were measured in the cell supernatant by an enzyme-linked immunosorbent assay (ELISA). The t-test method was used for the statistical analysis of the mean difference between groups. Results: EFTUD2 mRNA and protein expression levels were significantly increased in HepAD38 and HepG2-NTCP cells following AM679 treatment, with a statistically significant difference (P < 0.001). Intra-and extracellular indicators such as HBV DNA, HBV RNAs, HBV 3.5kb-RNA, HBsAg, and HBeAg were decreased to varying degrees in both cell models, and the decrease in these indicators was more pronounced with the increase in AM679 concentration and prolonged treatment duration, while the combined use of AM679 and entecavir had a more significant antiviral effect. The HBV DNA inhibition rates in the supernatant of HepAD38 cells with the use of 2 nmol/L AM679 were 21% and 48% on days three and nine, respectively. The AM679 combined with the ETV treatment group had the most significant inhibitory effect (62%), with a P < 0.01. More active HBV replication was observed after silencing EFTUD2, while the antiviral activity of AM679 was significantly weakened. Conclusion: AM679 exerts anti-HBV activity in vitro by targeting the regulation of EFTUD2 expression.

SARS-CoV-2 causes dysfunction in human iPSC-derived brainmicrovascular endothelial cells potentially by modulating the Wnt signaling pathway.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is associated with various neurological symptoms, including nausea, dizziness, headache, encephalitis, and epileptic seizures. SARS-CoV-2 is considered to affect the central nervous system (CNS) by interacting with the blood-brain barrier (BBB), which is defined by tight junctions that seal paracellular gaps between brain microvascular endothelial cells (BMECs). Although SARS-CoV-2 infection of BMECs has been reported, the detailed mechanism has not been fully elucidated. Using the original strain of SARS-CoV-2, the infection in BMECs was confirmed by a detection of intracellular RNA copy number and localization of viral particles. BMEC functions were evaluated by measuring transendothelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics, and expression levels of proinflammatory genes. BMEC signaling pathway was examined by comprehensive RNA-seq analysis. We observed that iPSC derived brain microvascular endothelial like cells (iPSC-BMELCs) were infected with SARS-CoV-2. SARS-CoV-2 infection resulted in decreased TEER. In addition, SARS-CoV-2 infection decreased expression levels of tight junction markers CLDN3 and CLDN11. SARS-CoV-2 infection also increased expression levels of proinflammatory genes, which are known to be elevated in patients with COVID-19. Furthermore, RNA-seq analysis revealed that SARS-CoV-2 dysregulated the canonical Wnt signaling pathway in iPSC-BMELCs. Modulation of the Wnt signaling by CHIR99021 partially inhibited the infection and the subsequent inflammatory responses. These findings suggest that SARS-CoV-2 infection causes BBB dysfunction via Wnt signaling. Thus, iPSC-BMELCs are a useful in vitro model for elucidating COVID-19 neuropathology and drug development.