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Amaryllidaceae alkaloids (AAs) are a diverse group of alkaloids exclusively reported from the Amaryllidaceae plant family. In planta, their biosynthesis is still not fully characterized, however, a labeling study established 4'-O-methylnorbelladine as the key intermediate compound of the pathway. Previous reports have characterized O-methyltransferases from several Amaryllidaceae species. Nevertheless, the formation of the different O-methylnorbelladine derivatives (3'-O-methylnorbelladine, 4'-O-methylnorbelladine, and 3'4'-O-dimethylnorbelladine), the role, and the preferred substrates of O-methyltransferases are not clearly understood. In this study, we performed the biochemical characterization of an O-methyltransferase candidate from Narcissus papyraceus (NpOMT) in vitro and in vivo, following biotransformation of norbelladine in Nicotiana benthamiana having transient expression of NpOMT. Docking analysis was further used to investigate substrate preferences, as well as key interacting residues of NpOMT. Our study shows that NpOMT methylates norbelladine preferentially at the 4'-OH position in vitro and in planta. Interestingly, NpOMT also catalyzed the synthesis of 3',4'-O-dimethylnorbelladine from norbelladine and 4'-O-methylnorbelladine during in vitro enzymatic assay. Furthermore, we show that NpOMT methylates 3,4-dihydroxybenzylaldehyde and caffeic acid in a non-regiospecific manner to produce meta/para monomethylated products. This study reveals a novel catalytic potential of an Amaryllidaceae O-methyltransferase and its ability to regioselectively methylate norbelladine in the heterologous host Nicotiana benthamiana.
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Covering: 2017 to 2023 (now)Amaryllidaceae alkaloids (AAs) are a unique class of specialized metabolites containing heterocyclic nitrogen bridging that play a distinct role in higher plants. Irrespective of their diverse structures, most AAs are biosynthesized via intramolecular oxidative coupling. The complex organization of biosynthetic pathways is constantly enlightened by new insights owing to the advancement of natural product chemistry, synthetic organic chemistry, biochemistry, systems and synthetic biology tools and applications. These promote novel compound identification, trace-level metabolite quantification, synthesis, and characterization of enzymes engaged in AA catalysis, enabling the recognition of biosynthetic pathways. A complete understanding of the pathway benefits biotechnological applications in the long run. This review emphasizes the structural diversity of the AA specialized metabolites involved in biogenesis although the process is not entirely defined yet. Moreover, this work underscores the pivotal role of synthetic and enantioselective studies in justifying biosynthetic conclusions. Their prospective candidacy as lead constituents for antiviral drug discovery has also been established. However, a complete understanding of the pathway requires further interdisciplinary efforts in which antiviral studies address the structure-activity relationship. This review presents current knowledge on the topic.
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Alcaloides de Amaryllidaceae , Antivirais , Antivirais/farmacologia , Antivirais/química , Antivirais/metabolismo , Alcaloides de Amaryllidaceae/farmacologia , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/metabolismo , Vias Biossintéticas , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Human muscle-specific RING fingers (MURFs) are members of the tripartite motif (TRIM) family of proteins characterized by their C-terminal subgroup one signature domain. MURFs play a role in sarcomere formation and microtubule dynamics. It was previously established that some TRIMs undergo post-translational modification by small ubiquitin-like modifier (SUMO). In this study, we explored the putative SUMOylation of MURF proteins as well as their interactions with SUMO. MURF proteins (TRIM54, TRIM55, and TRIM63) were not found to be SUMOylated. However, TRIM55 turnover by proteasomal and lysosomal degradation was higher upon overexpression of SUMO-3 but not of SUMO-1. Furthermore, it is predicted that TRIM55 contains two potential SUMO-interacting motifs (SIMs). We found that SIM1- and SIM2-mutated TRIM55 were more stable than the wild-type (WT) protein partly due to decreased degradation. Consistently, SIM-mutated TRIM55 was less polyubiquitinated than the WT protein, despite similar monoubiquitination levels. Using IF microscopy, we observed that SIM motifs influenced TRIM55 subcellular localization. In conclusion, our results suggest that SUMO-3 or SUMO-3-modified proteins modulate the localization, stability, and RING ubiquitin ligase activity of TRIM55.
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Proteína SUMO-1 , Ubiquitina , Humanos , Ubiquitina/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Amaryllidaceae alkaloid (AAs) biosynthesis has garnered significant attention in recent years, particularly with the commercialisation of galanthamine as a treatment for the symptoms of Alzheimer's disease. A significant amount of research work over the last 8 decades has focused on the understanding of AA biosynthesis, starting from early radiolabelling studies to recent multi-omics analysis with modern biotechnological advancements. Those studies enabled the identification of hundreds of metabolites, the characterisation of biochemical pathway, an understanding of the environmental stimuli, and of the molecular regulation of these pharmaceutically and agriculturally important metabolites. Despite the numerous works there remain significant gaps in understanding their biosynthesis in Amaryllidaceae plants. As such, further research is needed to fully elucidate the metabolic pathway and facilitate their production. This review aims to provide a comprehensive overall summary of the current state of knowledge on AAs biosynthesis, from elicitation of transcription factors expression in the cell nucleus to alkaloid transport in the apoplast, and to highlight the challenges that need to be overcome for further advancement.
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Dengue fever is an infectious disease caused by the dengue virus (DENV), an RNA Flavivirus transmitted by the mosquitoes Aedes aegypti and Aedes albopictus widespread in tropical, subtropical and also temperate regions. Symptoms range from a simple cold to a severe, life-threatening haemorrhagic fever. According to the WHO, it affects around 390 million people per year. No antiviral treatment for DENV is available, and the Dengvaxia vaccine is only intended for people over 9 years of age who have contracted dengue one time in the past, and shows serotype-specific effectiveness. There is therefore a crying need to discover new molecules with antiviral power against flaviviruses. The present study was carried out to evaluate the anti-DENV activities and cytotoxicity of triazenes obtained by diazocopulation. Some triazenes were highly cytotoxic (16, and 25) to hepatocarcinoma Huh7 cells, whereas others displayed strong anti-DENV potential. The antiviral activity ranged from EC50 = 7.82 µM to 48.12 µM in cellulo, with a selectivity index (CC50/EC50) greater than 9 for two of the compounds (10, and 20). In conclusion, these new triazenes could serve as a lead to develop and optimize drugs against DENV.
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Aedes , Vírus da Dengue , Dengue , Animais , Humanos , Dengue/tratamento farmacológico , Antivirais/farmacologiaRESUMO
The increasing demand for novel natural compounds has prompted the exploration of innovative approaches in bioengineering. This study investigates the bioengineering potential of the marine diatom Phaeodactylum tricornutum through the introduction of cannabis genes, specifically, tetraketide synthase (TKS), and olivetolic acid cyclase (OAC), for the production of the cannabinoid precursor, olivetolic acid (OA). P. tricornutum is a promising biotechnological platform due to its fast growth rate, amenability to genetic manipulation, and ability to produce valuable compounds. Through genetic engineering techniques, we successfully integrated the cannabis genes TKS and OAC into the diatom. P. tricornutum transconjugants expressing these genes showed the production of the recombinant TKS and OAC enzymes, detected via Western blot analysis, and the production of cannabinoids precursor (OA) detected using the HPLC/UV spectrum when compared to the wild-type strain. Quantitative analysis revealed significant olivetolic acid accumulation (0.6-2.6 mg/L), demonstrating the successful integration and functionality of the heterologous genes. Furthermore, the introduction of TKS and OAC genes led to the synthesis of novel molecules, potentially expanding the repertoire of bioactive compounds accessible through diatom-based biotechnology. This study demonstrates the successful bioengineering of P. tricornutum with cannabis genes, enabling the production of OA as a precursor for cannabinoid production and the synthesis of novel molecules with potential pharmaceutical applications.
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Canabinoides , Cannabis , Diatomáceas , Alucinógenos , Cannabis/genética , Canabinoides/genética , Diatomáceas/genética , Agonistas de Receptores de Canabinoides , BioengenhariaRESUMO
Amaryllidaceae alkaloids (AAs) are a structurally diverse family of alkaloids recognized for their many therapeutic properties, such as antiviral, anti-cholinesterase, and anticancer properties. Norbelladine and its derivatives, whose biological properties are poorly studied, are key intermediates required for the biosynthesis of all ~650 reported AAs. To gain insight into their therapeutic potential, we synthesized a series of O-methylated norbelladine-type alkaloids and evaluated their cytotoxic effects on two types of cancer cell lines, their antiviral effects against the dengue virus (DENV) and the human immunodeficiency virus 1 (HIV-1), and their anti-Alzheimer's disease (anti-cholinesterase and -prolyl oligopeptidase) properties. In monocytic leukemia cells, norcraugsodine was highly cytotoxic (CC50 = 27.0 µM), while norbelladine was the most cytotoxic to hepatocarcinoma cells (CC50 = 72.6 µM). HIV-1 infection was impaired only at cytotoxic concentrations of the compounds. The 3,4-dihydroxybenzaldehyde (selectivity index (SI) = 7.2), 3',4'-O-dimethylnorbelladine (SI = 4.8), 4'-O-methylnorbelladine (SI > 4.9), 3'-O-methylnorbelladine (SI > 4.5), and norcraugsodine (SI = 3.2) reduced the number of DENV-infected cells with EC50 values ranging from 24.1 to 44.9 µM. The O-methylation of norcraugsodine abolished its anti-DENV potential. Norbelladine and its O-methylated forms also displayed butyrylcholinesterase-inhibition properties (IC50 values ranging from 26.1 to 91.6 µM). Altogether, the results provided hints of the structure−activity relationship of norbelladine-type alkaloids, which is important knowledge for the development of new inhibitors of DENV and butyrylcholinesterase.
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Alcaloides , Alcaloides de Amaryllidaceae , Amaryllidaceae , Alcaloides/química , Alcaloides/farmacologia , Amaryllidaceae/metabolismo , Alcaloides de Amaryllidaceae/química , Antivirais/farmacologia , Butirilcolinesterase , Inibidores da Colinesterase , Humanos , Tiramina/análogos & derivadosRESUMO
Dengue fever, caused by dengue virus (DENV), is the most prevalent arthropod-borne viral disease and is endemic in many tropical and subtropical parts of the world, with an increasing incidence in temperate regions. The closely related flavivirus Zika virus (ZIKV) can be transmitted vertically in utero and causes congenital Zika syndrome and other birth defects. In adults, ZIKV is associated with Guillain-Barré syndrome. There are no approved antiviral therapies against either virus. Effective antiviral compounds are urgently needed. Amaryllidaceae alkaloids (AAs) are a specific class of nitrogen-containing compounds produced by plants of the Amaryllidaceae family with numerous biological activities. Recently, the AA lycorine was shown to present strong antiflaviviral properties. Previously, we demonstrated that Crinum jagus contained lycorine and several alkaloids of the cherylline, crinine, and galanthamine types with unknown antiviral potential. In this study, we explored their biological activities. We show that C. jagus crude alkaloid extract inhibited DENV infection. Among the purified AAs, cherylline efficiently inhibited both DENV (50% effective concentration [EC50], 8.8 µM) and ZIKV replication (EC50, 20.3 µM) but had no effect on HIV-1 infection. Time-of-drug-addition and -removal experiments identified a postentry step as the one targeted by cherylline. Consistently, using subgenomic replicons and replication-defective genomes, we demonstrate that cherylline specifically hinders the viral RNA synthesis step but not viral translation. In conclusion, AAs are an underestimated source of antiflavivirus compounds, including the effective inhibitor cherylline, which could be optimized for new therapeutic approaches.
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Alcaloides , Alcaloides de Amaryllidaceae , Amaryllidaceae , Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Adulto , Alcaloides/farmacologia , Alcaloides de Amaryllidaceae/farmacologia , Humanos , Isoquinolinas , Replicação Viral , Infecção por Zika virus/tratamento farmacológicoRESUMO
Amaryllidaceae plants are rich in alkaloids with biological properties. Pancratium trianthum is an Amaryllidaceae species widely used in African folk medicine to treat several diseases such as central nervous system disorders, tumors, and microbial infections, and it is used to heal wounds. The current investigation explored the biological properties of alkaloid extracts from bulbs of P. trianthum collected in the Senegalese flora. Alkaloid extracts were analyzed and identified by chromatography and mass spectrometry. Alkaloid extracts from P. trianthum displayed pleiotropic biological properties. Cytotoxic activity of the extracts was determined on hepatocarcinoma Huh7 cells and on acute monocytic leukemia THP-1 cells, while agar diffusion and microdilution assays were used to evaluate antibacterial activity. Antiviral activity was measured by infection of extract-treated cells with dengue virus (DENVGFP) and human immunodeficiency virus-1 (HIV-1GFP) reporter vectors. Cytotoxicity and viral inhibition were the most striking of P. trianthum's extract activities. Importantly, non-cytotoxic concentrations were highly effective in completely preventing DENVGFP replication and in reducing pseudotyped HIV-1GFP infection levels. Our results show that P. trianthum is a rich source of molecules for the potential discovery of new treatments against various diseases. Herein, we provide scientific evidence to rationalize the traditional uses of P. trianthum for wound treatment as an anti-dermatosis and antiseptic agent.
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Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/farmacologia , Amaryllidaceae/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Dengue/tratamento farmacológico , Vírus da Dengue/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
Elite controllers (ECs) are a rare subset of HIV-1 slow progressors characterized by prolonged viremia suppression. HLA alleles B27 and B57 promote the cytotoxic T lymphocyte (CTL)-mediated depletion of infected cells in ECs, leading to the emergence of escape mutations in the viral capsid (CA). Whether those mutations modulate CA detection by innate sensors and effectors is poorly known. Here, we investigated the targeting of CA from B27/B57+ individuals by cytosolic antiviral factors Mx2 and TRIM5α. Toward that aim, we constructed chimeric HIV-1 vectors using CA isolated from B27/B57+ or control subjects. HIV-1 vectors containing B27/B57+-specific CA had increased sensitivity to TRIM5α but not to Mx2. Following exposure to those vectors, cells showed increased resistance against both TRIM5α-sensitive and -insensitive HIV-1 strains. Induction of the antiviral state did not require productive infection by the TRIM5α-sensitive virus, as shown using chemically inactivated virions. Depletion experiments revealed that TAK1 and Ubc13 were essential to the TRIM5α-dependent antiviral state. Accordingly, induction of the antiviral state was accompanied by the activation of NF-κB and AP-1 in THP-1 cells. Secretion of IFN-I was involved in the antiviral state in THP-1 cells, as shown using a receptor blocking antibody. This work identifies innate activation pathways that are likely to play a role in the natural resistance to HIV-1 progression in ECs.
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Proteínas de Transporte/metabolismo , HIV-1/genética , Proteínas de Resistência a Myxovirus/metabolismo , Adulto , Antivirais , Fatores de Restrição Antivirais , Linfócitos T CD8-Positivos/imunologia , Capsídeo/metabolismo , Capsídeo/fisiologia , Epitopos de Linfócito T/imunologia , Feminino , Infecções por HIV/imunologia , Soropositividade para HIV , HIV-1/imunologia , Antígenos HLA-B/genética , Antígeno HLA-B27/genética , Humanos , Masculino , Linfócitos T Citotóxicos/imunologia , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Viremia , Replicação Viral/imunologiaRESUMO
BACKGROUND: The fight against the COVID-19 pandemic has created an urgent need to rapidly detect infected people. The challenge for clinical laboratories has been finding a high throughput, cost-efficient, and accurate testing method in the context of extraction reagents shortage on a global scale. To answer this need, we studied SARS-CoV-2 detection in oro-nasopharyngeal (ONP) swabs stored in Universal Transport Media (UTM) or in RNase-free water by rRT-PCR with Seegene Allplex™ 2019-nCoV assay without RNA extraction. RESULTS: Optimal results were obtained when swabs stored in UTM were diluted 1/5 and 1/2 in RNase-free water. Thermal lysis before rRT-PCR testing slightly improved detection rate. In addition, proteinase K (PK) treatment allowed for a significant reduction of invalid results and increased sensitivity for detection of low viral load specimens. In a panel of positive samples with all 3 viral genes amplified and N gene Cycle threshold values (Ct values) from 15 to 40, our detection rate was 98.9% with PK and 94.4% without. In a challenging panel of low positive samples with only the N gene being detectable at Ct values > 30, detection rate was increased from 53.3 to 76.7% with the addition of PK, and invalid rate fell off from 18.3 to 0%. Furthermore, we demonstrated that our method reliably detects specimens with Ct values up to 35, whereas false negative samples become frequent above this range. Finally, we show that swabs should be stored at - 70 °C rather than 4 °C when testing cannot be performed within 72 h of collection. CONCLUSION: We successfully optimized the unextracted rRT-PCR process using the Seegene Allplex™ 2019-nCoV assay to detect SARS-CoV-2 RNAs in nasopharyngeal swabs. This improved method offers cost savings and turnaround time advantages compared to automated extraction, with high efficiency of detection that could play an important role in the surveillance of Covid-19.
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Teste para COVID-19/métodos , COVID-19/virologia , Nasofaringe/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Teste para COVID-19/economia , Humanos , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/economia , Ribonucleases/química , Manejo de Espécimes/métodos , Carga ViralRESUMO
Alkaloids are an important group of specialized nitrogen metabolites with a wide range of biochemical and pharmacological effects. Since the first publication on lycorine in 1877, more than 650 alkaloids have been extracted from Amaryllidaceae bulbous plants and clustered together as the Amaryllidaceae alkaloids (AAs) family. AAs are specifically remarkable for their diverse pharmaceutical properties, as exemplified by the success of galantamine used to treat the symptoms of Alzheimer's disease. This review addresses the isolation, biological, and structure activity of AAs discovered from January 2015 to August 2020, supporting their therapeutic interest.
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Alcaloides de Amaryllidaceae/metabolismo , Alcaloides de Amaryllidaceae/farmacologia , Descoberta de Drogas , Alcaloides de Amaryllidaceae/química , Animais , HumanosRESUMO
In this review, we summarize recent advances in the knowledge of the biological functions of human TRIM5α, a cytoplasmic protein mostly known for its antiretroviral functions. In addition to directly targeting retroviral capsid cores, an inhibitory activity called "restriction", TRIM5α senses retroviruses and activates NF-κB and AP-1 signaling pathways, resulting in the production of type I interferon (IFN-I). The antiviral state resulting from the activation of these pathways includes the upregulation of other restriction factors, and is thought to be important for the control of HIV-1 in some patients. TRIM5α also targets the protease enzyme of several tick-borne flaviviruses, a family of viruses not closely related to retroviruses. In addition to these antiviral functions, TRIM5α promotes autophagy by interacting with key actors of this pathway, such as ULK1 and p62. TRIM5α may function as a selective autophagy receptor in some conditions. Altogether, our understanding of TRIM5α shows its potential for the development of medical applications in viral diseases and beyond.
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Antivirais , HIV-1 , Fatores de Restrição Antivirais , Capsídeo , Proteínas de Transporte/genética , HIV-1/genética , Humanos , Retroviridae , Proteínas com Motivo Tripartido , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: The promyelocytic leukemia (PML) protein, a type I interferon (IFN-I)-induced gene product and a member of the tripartite motif (TRIM) family, modulates the transcriptional activity of viruses belonging to various families. Whether PML has an impact on the replication of HIV-1 has not been fully addressed, but recent studies point to its possible involvement in the restriction of HIV-1 in human cells and in the maintenance of transcriptional latency in human cell lines in which HIV-1 is constitutively repressed. We investigated further the restriction of HIV-1 and a related lentivirus, SIVmac, by PML in murine cells and in a lymphocytic human cell line. In particular, we studied the relevance of PML to IFN-I-mediated inhibition and the role of individual human isoforms. RESULTS: We demonstrate that both human PML (hPML) and murine PML (mPML) inhibit the early post-entry stages of the replication of HIV-1 and a related lentivirus, SIVmac. In addition, HIV-1 was transcriptionally silenced by mPML and by hPML isoforms I, II, IV and VI in MEFs. This PML-mediated transcriptional repression was attenuated in presence of the histone deacetylase inhibitor SAHA. In contrast, depletion of PML had no effect on HIV-1 gene expression in a human T cell line. PML was found to contribute to the inhibition of HIV-1 by IFN-I. Specifically, IFN-α and IFN-ß treatments of MEFs enhanced the PML-dependent inhibition of HIV-1 early replication stages. CONCLUSIONS: We show that PML can inhibit HIV-1 and other lentiviruses as part of the IFN-I-mediated response. The restriction takes place at two distinct steps, i.e. reverse transcription and transcription, and in an isoform-specific, cellular context-specific fashion. Our results support a model in which PML activates innate immune antilentiviral effectors. These data are relevant to the development of latency reversal-inducing pharmacological agents, since PML was previously proposed as a pharmacological target for such inhibitors. This study also has implications for the development of murine models of HIV-1.
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HIV-1/imunologia , Interações Hospedeiro-Patógeno , Proteínas Nucleares/metabolismo , Vírus da Imunodeficiência Símia/imunologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Replicação Viral , Animais , Linhagem Celular , HIV-1/fisiologia , Humanos , Interferon Tipo I/metabolismo , Camundongos , Dados de Sequência Molecular , Proteína da Leucemia Promielocítica , Transcrição Reversa , Análise de Sequência de DNA , Vírus da Imunodeficiência Símia/fisiologia , Transcrição GênicaRESUMO
CMV and varicella zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplantation (UCBT). However, the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. In this study, the reconstitution of CMV- and VZV-specific T cell responses was assessed using IFN-γ ELISPOT in 28 children who underwent UCBT to treat hematological or inherited disorders. Barely detectable in the first 3 mo posttransplantation, CMV- and VZV-specific T cell responses were observed in 30.4% and 40.3% of study subjects after 36 mo of follow-up. Four of five CMV-seropositive subjects developed detectable levels of circulating CMV DNA (DNAemia), and 5 of 17 VZV-seropositive patients experienced herpes zoster during the posttransplant period. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and four subjects developed a VZV-specific IFN-γ response without clinical signs of infection. No CMV- or VZV-related events were observed in study subjects following the development of CMV- or VZV-specific responses > 150 spot-forming units/10(6) PBMCs, consistent with T cell-mediated protection. Finally, famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire, because the frequency of T cells producing IFN-γ in response to VZV Ags reached levels consistent with protection in two nonzoster subjects. Monitoring of CMV- and VZV-specific cell-mediated immunity could inform immunocompetence and guide the initiation and cessation of antiherpetic prophylaxis in UCBT recipients.
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Varicela/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Herpesvirus Humano 3/imunologia , Recuperação de Função Fisiológica/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Criança , Pré-Escolar , ELISPOT/métodos , Feminino , Seguimentos , Doenças Genéticas Inatas/imunologia , Doenças Genéticas Inatas/terapia , Doenças Hematológicas/imunologia , Doenças Hematológicas/terapia , Humanos , Lactente , Interferon gama/imunologia , Masculino , Transplante HomólogoRESUMO
Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.
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Genes Reporter , Replicação do RNA , RNA Viral , Replicon , SARS-CoV-2 , Humanos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Cromossomos Artificiais Bacterianos/genética , COVID-19/virologia , COVID-19/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Regiões Promotoras Genéticas , Replicon/genética , Replicação do RNA/genética , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+ ) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform.
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Chlamydomonas reinhardtii , Inibidores de Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Vorinostat , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/química , Histona Desacetilases/metabolismo , Transgenes/genéticaRESUMO
Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3'-hydroxylase (LaeC3'H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3'H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.
Assuntos
Alcaloides de Amaryllidaceae , Proteínas de Plantas , Transcinamato 4-Mono-Oxigenase , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Transcinamato 4-Mono-Oxigenase/metabolismo , Transcinamato 4-Mono-Oxigenase/genética , Alcaloides de Amaryllidaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Ácidos Cumáricos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Recipients of umbilical cord blood (UCB) transplantation (UCBT) face a high risk of morbidity and mortality related to opportunistic infections (OI) and leukemic relapse. To understand the molecular basis of these UCBT-related complications, the characteristics of UCB-derived antigen-specific CD8(+) T cells were examined in a group of pediatric UCBT recipients. Compared with the UCB graft inoculum and the late post-UCBT period (12-36 months), declining clonal diversity of UCB-derived CD8(+) T cells specific for the Melan-A(26-35) A27L peptide and high frequencies of PD-1-expressing CD8(+) T cells were observed in the first 3 months after UCBT, a period during which OIs are most frequent. The CD8(+) T-cell compartment predominantly comprised CD45RA(+) CCR7(-) terminally differentiated effector-memory T cells until 6 months after UCBT, at which time the polyfunctionality of antigen-specific CD8(+) T cells was reestablished. Finally, the frequency of PD-1(+) CD8(+) T cells was significantly higher in subjects who subsequently experienced leukemic relapse. This study informs the biologic properties of UCB-derived CD8(+) T cells and provides a rationale for the characteristics of UCBT in terms of immune reconstitution and OI. These results also suggest that the elevated frequency of PD-1(+) CD8(+) T cells could be associated with leukemic relapse in pediatric UCBT recipients.