RESUMO
Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , COVID-19 , Pirazóis , Quinolinas , Humanos , SARS-CoV-2/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Simulação de Acoplamento Molecular , Tratamento Farmacológico da COVID-19 , Antivirais/químicaRESUMO
BACKGROUND: Respiratory infections have long been recognized as a primary cause of acute exacerbation of chronic obstructive pulmonary disease (AE-COPD). Additionally, the emergence of antimicrobial resistance has led to an urgent and critical situation in developing countries, including Vietnam. This study aimed to investigate the distribution and antimicrobial resistance of bacteria in patients with AE-COPD using both conventional culture and multiplex real-time PCR. Additionally, associations between clinical characteristics and indicators of pneumonia in these patients were examined. METHODS: This cross-sectional prospective study included 92 AE-COPD patients with pneumonia and 46 without pneumonia. Sputum specimens were cultured and examined for bacterial identification, and antimicrobial susceptibility was determined for each isolate. Multiplex real-time PCR was also performed to detect ten bacteria and seven viruses. RESULTS: The detection rates of pathogens in AE-COPD patients with pneumonia were 92.39%, compared to 86.96% in those without pneumonia. A total of 26 pathogenic species were identified, showing no significant difference in distribution between the two groups. The predominant bacteria included Klebsiella pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae, followed by Acinetobacter baumannii and Streptococcus mitis. There was a slight difference in antibiotic resistance between bacteria isolated from two groups. The frequency of H. influenzae was notably greater in AE-COPD patients who experienced respiratory failure (21.92%) than in those who did not (9.23%). S. pneumoniae was more common in patients with stage I (44.44%) or IV (36.36%) COPD than in patients with stage II (17.39%) or III (9.72%) disease. ROC curve analysis revealed that C-reactive protein (CRP) levels could distinguish patients with AE-COPD with and without pneumonia (AUC = 0.78). CONCLUSION: Gram-negative bacteria still play a key role in the etiology of AE-COPD patients, regardless of the presence of pneumonia. This study provides updated evidence for the epidemiology of AE-COPD pathogens and the appropriate selection of antimicrobial agents in Vietnam.
Assuntos
Antibacterianos , Bactérias , Farmacorresistência Bacteriana , Doença Pulmonar Obstrutiva Crônica , Humanos , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/microbiologia , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Estudos Transversais , Vietnã/epidemiologia , Estudos Prospectivos , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias/isolamento & purificação , Bactérias/efeitos dos fármacos , Bactérias/classificação , Bactérias/genética , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/epidemiologia , Testes de Sensibilidade Microbiana , Escarro/microbiologia , Idoso de 80 Anos ou mais , Pneumonia/microbiologia , Pneumonia/tratamento farmacológico , Pneumonia/epidemiologiaRESUMO
While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.
Assuntos
Ácido Clorogênico/análogos & derivados , Enterovirus Humano A , Infecções por Enterovirus , Ilex , Plantas Medicinais , Antivirais/uso terapêutico , Linhagem Celular Tumoral , Ácido Clorogênico/uso terapêutico , Enterovirus Humano A/genética , Infecções por Enterovirus/tratamento farmacológico , Heparitina Sulfato/metabolismo , Humanos , Ilex/química , Simulação de Acoplamento Molecular , Extratos Vegetais/uso terapêutico , Plantas Medicinais/químicaRESUMO
Dried Iris rhizomes have been used in Chinese and European traditional medicine for the treatment of various diseases such as bacterial infections, cancer, and inflammation, as well as for being astringent, laxative, and diuretic agents. Eighteen phenolic compounds including some rare secondary metabolites, such as irisolidone, kikkalidone, irigenin, irisolone, germanaism B, kaempferol, and xanthone mangiferin, were isolated for the first time from Iris aphylla rhizomes. The hydroethanolic Iris aphylla extract and some of its isolated constituents showed protective effects against influenza H1N1 and enterovirus D68 and anti-inflammatory activity in human neutrophils. The promising anti-influenza effect of apigenin (13: , almost 100% inhibition at 50 µM), kaempferol (14: , 92%), and quercetin (15: , 48%) were further confirmed by neuraminidase inhibitory assay. Irisolidone (1: , almost 100% inhibition at 50 µM), kikkalidone (5: , 93%), and kaempferol (14: , 83%) showed promising anti-enterovirus D68 activity in vitro. The identified compounds were plotted using ChemGPS-NP to correlate the observed activity of the isolated phenolic compounds with the in-house database of anti-influenza and anti-enterovirus agents. Our results indicated that the hydroethanolic Iris aphylla extract and Iris phenolics hold the potential to be developed for the management of seasonal pandemics of influenza and enterovirus infections.
Assuntos
Flavonas , Vírus da Influenza A Subtipo H1N1 , Gênero Iris , Humanos , Quempferóis , Extratos Vegetais/farmacologia , Rizoma/química , Antivirais/farmacologia , Relação Estrutura-Atividade , Fenóis/análise , Anti-Inflamatórios/farmacologiaRESUMO
In December 2020, the U.K. authorities reported to the World Health Organization (WHO) that a new COVID-19 variant, considered to be a variant under investigation from December 2020 (VUI-202012/01), was identified through viral genomic sequencing. Although several other mutants were previously reported, VUI-202012/01 proved to be about 70% more transmissible. Hence, the usefulness and effectiveness of the newly U.S. Food and Drug Administration (FDA)-approved COVID-19 vaccines against these new variants are doubtfully questioned. As a result of these unexpected mutants from COVID-19 and due to lack of time, much research interest is directed toward assessing secondary metabolites as potential candidates for developing lead pharmaceuticals. In this study, a marine-derived fungus Aspergillus terreus was investigated, affording two butenolide derivatives, butyrolactones I (1) and III (2), a meroterpenoid, terretonin (3), and 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde (4). Chemical structures were unambiguously determined based on mass spectrometry and extensive 1D/2D NMR analyses experiments. Compounds (1-4) were assessed for their in vitro anti-inflammatory, antiallergic, and in silico COVID-19 main protease (Mpro) and elastase inhibitory activities. Among the tested compounds, only 1 revealed significant activities comparable to or even more potent than respective standard drugs, which makes butyrolactone I (1) a potential lead entity for developing a new remedy to treat and/or control the currently devastating and deadly effects of COVID-19 pandemic and elastase-related inflammatory complications.
Assuntos
4-Butirolactona/análogos & derivados , Antialérgicos/química , Anti-Inflamatórios/química , Aspergillus/química , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/antagonistas & inibidores , 4-Butirolactona/química , 4-Butirolactona/isolamento & purificação , 4-Butirolactona/metabolismo , Antialérgicos/metabolismo , Anti-Inflamatórios/metabolismo , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Neutrófilos/enzimologia , SARS-CoV-2/isolamento & purificação , Água do Mar/microbiologia , Proteínas da Matriz Viral/metabolismoRESUMO
MicroRNAs are noncoding RNA species comprising 18-23 nucleotides that regulate host-virus interaction networks. Here, we show that enterovirus A71 infection in human rhabdomyosarcoma (RD) is regulated by miR-197 expression. Transfection of miR-197 mimic into RD cells inhibited virus replication by interfering with the viral RNA synthesis. We employed a combination of mass-spectrometry-based quantitative proteomics with the stable isotope labeling with amino acids in cell culture (SILAC) approach for the identification of the miR-197 target genes in RD cells and to investigate the differential expression of the prospective target proteins. A total of 1822 proteins were repeatedly identified in miR-197-transfected RD cells, 106 of which were predicted to have seed sites by TargetScan. Notably, seven of eight selected genes potentially related to viral replication and immune response were validated as direct miR-197 targets, using a luciferase 3'-untranslated region (UTR) reporter assay. The expression levels of three selected endogenous molecules (ITGAV, ETF1, and MAP2K1/MEK1) were significantly reduced when RD cells were transfected with a miR-197 mimic. Our results provide a comprehensive database of miR-197 targets, which might provide better insights into the understanding of host-virus interaction.
Assuntos
Enterovirus Humano A/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/fisiologia , Proteômica/métodos , Rabdomiossarcoma/virologia , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/farmacologia , RNA Viral/efeitos dos fármacos , Rabdomiossarcoma/genética , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Due to limited coding capacity of viral genome, enterovirus A71 (EV-A71) co-opts host nuclear proteins for its replication. Upon ER stress, the ER-localized 90 kDa activating transcription factor 6 (p90ATF6) is proteolytically cleaved to produce the transcriptionally active amino-terminal 50 kDa (p50ATF6) product where it enters the nucleus to activate a subset of unfolded protein response and ER-associated degradation (also known as ERAD) genes. During EV-A71 infection, however, this p50ATF6 product was not detected in the nucleus, and its downstream target genes were not activated. METHODS: We examined the role of ATF6 during EV-A71 infection, including its cleavage process and its role in viral life cycle by silencing or overexpressing ATF6. RESULTS: We showed that a potential cleavage in the middle of p90ATF6 produced an amino-terminal ~ 45 kDa fragment in a viral protease-independent but EV-A71-dependent manner. The disappearance of ATF6 was not restricted to a specific strain of EV-A71 or cell type, and was not simply caused by picornavirus-mediated global translational shutoff. This cleavage of ATF6, which was most likely mediated by the host response, was nevertheless independent of both cellular caspases and XBP1-associated proteasomes. The silencing of ATF6 expression by small interfering RNA suppressed viral titers due to reduced viral protein stability. This effect was markedly restored by the ectopic expression of p90ATF6. CONCLUSION: Our findings indicate that ATF6 plays a distinct role in viral protein stability and that the host uses different cleavage strategies, rather than conventional cleavage by generating p50ATF6, to combat viral infection.
Assuntos
Fator 6 Ativador da Transcrição/genética , Estresse do Retículo Endoplasmático , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/virologia , Transdução de Sinais , Proteínas Virais/química , Fator 6 Ativador da Transcrição/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Estabilidade ProteicaRESUMO
UNLABELLED: Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication. IMPORTANCE: Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.
Assuntos
Enterovirus Humano A/imunologia , Enterovirus Humano A/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Proteínas Virais/biossíntese , Replicação Viral , Proteína ran de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , HumanosRESUMO
OBJECTIVES: Enterovirus 71 (EV-A71) is an important pathogen that can cause severe neurological symptoms and even death. Our aim was to identify potent anti-EV-A71 compounds and study their underlying mechanisms and in vivo activity. METHODS: We identified a potent imidazolidinone derivative (abbreviated to PR66) as an inhibitor of EV-A71 infection from the screening of compounds and subsequent structure-based modification. Time-course treatments and resistant virus selection of PR66 were employed to study the mode of mechanism of PR66. In vivo activity of PR66 was tested in the ICR strain of new-born mice challenged with EV-A71/4643/MP4. RESULTS: PR66 could impede the uncoating process during viral infection via interaction with capsid protein VP1, as shown by a resistant virus selection assay. Using site-directed mutagenesis, we confirmed that a change from valine to phenylalanine in the 179th amino acid residue of the cDNA-derived resistant virus resulted in resistance to PR66. PR66 increased the virion stability of WT viruses, but not the PR66-resistant mutant, in a particle stability thermal release assay. We further showed that PR66 had excellent anti-EV-A71 activity in an in vivo mouse model of disease, with a dose-dependent increase in survival rate and in protection against virus-induced hind-limb paralysis following oral or intraperitoneal administration. This was associated with reductions of viral titres in brain and muscle tissues. CONCLUSIONS: We demonstrated here for the first time that an imidazolidinone derivative (PR66) could protect against EV-A71-induced neurological symptoms in vivo by suppressing EV-A71 replication. This involved binding to and restricting viral uncoating.
Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Enterovirus Humano A/efeitos dos fármacos , Animais , Antivirais/isolamento & purificação , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Humanos , Concentração Inibidora 50 , Camundongos Endogâmicos ICR , Análise de SobrevidaRESUMO
The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>10(5) copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5' untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.
Assuntos
Regiões 5' não Traduzidas , Enterovirus Humano A/genética , Regulação Viral da Expressão Gênica , Biossíntese de Proteínas , Pequeno RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Ribonuclease III/metabolismo , Proteínas Virais/biossínteseRESUMO
The non-structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α-helical domains (AH1 and AH2). Its self-oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host-encoded ER-associated reticulon 3 (RTN3) protein is involved in the formation of the replication-associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self-interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA-transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.
Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/imunologia , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Células Cultivadas , Hepatócitos/virologia , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de ProteínasRESUMO
Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Hepacivirus/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia , Regulação Alostérica , Antivirais/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Genótipo , Células HEK293 , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , RNA Interferente Pequeno/genética , Replicon , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genéticaRESUMO
Far-upstream element-binding protein 2 (FBP2) is an internal ribosomal entry site (IRES) trans-acting factor (ITAF) that negatively regulates enterovirus 71 (EV71) translation. This study shows that EV71 infection cleaved FBP2. Live EV71 and the EV71 replicon (but not UV-inactivated virus particles) induced FBP2 cleavage, suggesting that viral replication results in FBP2 cleavage. The results also showed that virus-induced proteasome, autophagy, and caspase activity co-contribute to EV71-induced FBP2 cleavage. Using FLAG-fused FBP2, we mapped the potential cleavage fragments of FBP2 in infected cells. We also found that FBP2 altered its function when its carboxyl terminus was cleaved. This study presents a mechanism for virus-induced cellular events to cleave a negative regulator for viral IRES-driven translation.
Assuntos
Enterovirus Humano A/metabolismo , Infecções por Enterovirus/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Internalização do Vírus , Western Blotting , Linhagem Celular Tumoral , Humanos , Plasmídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , Replicação Viral/fisiologiaRESUMO
Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.
Assuntos
Antivirais , Vírus da Influenza A , Extratos Vegetais , Antivirais/farmacologia , Antivirais/química , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Humanos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Animais , Células Madin Darby de Rim Canino , Cães , Internalização do Vírus/efeitos dos fármacos , Sapindaceae/química , Replicação Viral/efeitos dos fármacos , Ligação Viral/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Neuraminidase/metabolismo , Células A549 , Linhagem CelularRESUMO
In Indonesia, malaria remains a problem, with 94,610 active cases in 2021 and its current therapy includes chloroquine and artemisinin; however, resistance has been commonly reported. To overcome this problem, studies about potential medicinal plants that can be used as antimalaria, such as moringa (Moringa oleifera) started to receive more attention. The aim of this study was to investigate the effects of moringa in parasitemia, monocyte activation, and organomegaly on animal model malaria. This experimental study used male Mus musculus, infected by Plasmodium berghei ANKA, as an animal malaria model. The extract was made by maceration of dry moringa leaves, which were then divided into three concentrations: 25%, 50%, and 75%. Dihydroartemisinin-piperazine was used as a positive control treatment, and distilled water as a negative control treatment. The animals were observed for six days to assess the parasitemia count and the number of monocyte activation. On day 7, the animals were terminated, and the liver, spleen, and kidney were weighed. The results showed that the effective concentrations in reducing parasitemia and inducing monocyte activation were 50% and 25% of moringa leaf extract, respectively. The smallest liver and spleen enlargement was observed among animals within the group treated with a 50% concentration of M. oleifera extract. In contrast, the smallest kidney enlargement was observed in the group treated with 25% of M. oleifera extract. Further analysis is recommended to isolate compounds with antimalarial properties in moringa leaves.
Assuntos
Modelos Animais de Doenças , Malária , Monócitos , Parasitemia , Extratos Vegetais , Plasmodium berghei , Animais , Camundongos , Plasmodium berghei/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Masculino , Malária/tratamento farmacológico , Malária/parasitologia , Malária/imunologia , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Monócitos/imunologia , Parasitemia/tratamento farmacológico , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Moringa/química , Moringa oleifera/química , Folhas de Planta/química , Baço/efeitos dos fármacos , Baço/parasitologia , Baço/patologia , Baço/imunologia , Tamanho do Órgão/efeitos dos fármacosRESUMO
Influenza A viruses spread out worldwide, causing several global concerns. Hence, discovering neuraminidase inhibitors to prevent the influenza A virus is of great interest. In this work, a machine learning model was employed to evaluate the ligand-binding affinity of ca. 10 000 compounds from the MedChemExpress (MCE) database for inhibiting neuraminidase. Atomistic simulations, including molecular docking and molecular dynamics simulations, then confirmed the ligand-binding affinity. Furthermore, we clarified the physical insights into the binding process of ligands to neuraminidase. It was found that five compounds, including micronomicin, didesmethyl cariprazine, argatroban, Kgp-IN-1, and AY 9944, are able to inhibit neuraminidase N1 of the influenza A virus. Ten residues, including Glu119, Asp151, Arg152, Trp179, Gln228, Glu277, Glu278, Arg293, Asn295, and Tyr402, may be very important in controlling the ligand-binding process to N1.
RESUMO
VP39, an essential 2'-O-RNA methyltransferase enzyme discovered in Monkeypox virus (MPXV), plays a vital role in viral RNA replication and transcription. Inhibition of the enzyme may prevent viral replication. In this context, using a combination of molecular docking and molecular dynamics (MDs) simulations, the inhibitory ability of NCI Diversity Set VII natural compounds to VP39 protein was investigated. It should be noted that the computed binding free energy of ligand via molecular docking and linear interaction energy (LIE) approaches are in good agreement with the corresponding experiments with coefficients of R=0.72 and 0.75, respectively. NSC 319990, NSC 196515 and NSC 376254 compounds were demonstrated that can inhibit MPVX methyltransferase VP39 protein with the similar affinity compared to available inhibitor sinefungin. Moreover, nine residues involving Gln39, Gly68, Gly72, Asp95, Arg97, Val116, Asp138, Arg140 and Asn156 may be argued that they play an important role in binding process of inhibitors to VP39.Communicated by Ramaswamy H. Sarma.
RESUMO
Since December 2019, the Coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has spread rapidly around the world, overburdening healthcare systems and creating significant global health concerns. Rapid detection of infected individuals via early diagnostic tests and administration of effective therapy remains vital in pandemic control, and recent advances in the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system may support the development of novel diagnostic and therapeutic approaches. Cas-based SARS-CoV-2 detection methods (FnCAS9 Editor Linked Uniform Detection Assay (FELUDA), DNA endonuclease-targeted CRISPR trans reporter (DETECTR), and Specific High-sensitivity Enzymatic Reporter Unlocking (SHERLOCK)) have been developed for easier handling compared to quantitative polymerase chain reaction (qPCR) assays, with good rapidity, high specificity, and reduced need for complex instrumentation. Cas-CRISPR-derived RNA (Cas-crRNA) complexes have been shown to reduce viral loads in the lungs of infected hamsters, by degrading virus genomes and limiting viral replication in host cells. Viral-host interaction screening platforms have been developed using the CRISPR-based system to identify essential cellular factors involved in pathogenesis, and CRISPR knockout (CRISPRKO) and activation screening results have revealed vital pathways in the life cycle of coronaviruses, including host cell entry receptors (ACE2, DPP4, and ANPEP), proteases involved in spike activation and membrane fusion (cathepsin L (CTSL) and transmembrane protease serine 2 (TMPRSS2)), intracellular traffic control routes for virus uncoating and budding, and membrane recruitment for viral replication. Several novel genes (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin, subfamily A, member 4 (SMARCA4), ARIDIA, and KDM6A) have also been identified via systematic data mining analysis as pathogenic factors for severe CoV infection. This review highlights how CRISPR-based systems can be applied to investigate the viral life cycle, detect viral genomes, and develop therapies against SARS-CoV-2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Interações entre Hospedeiro e Microrganismos , Pandemias , Pulmão , Teste para COVID-19 , DNA Helicases , Proteínas Nucleares , Fatores de TranscriçãoRESUMO
The aim of this study was to identify the antiviral mechanism of a novel compound, BPR3P0128. From a large-scale screening of a library of small compounds, BPR3P compounds were found to be potent inhibitors of influenza viral replication in Madin-Darby canine kidney (MDCK) cells. BPR3P0128 exhibited inhibitory activity against both influenza A and B viruses. The 50% inhibitory concentrations were in the range of 51 to 190 nM in MDCK cells, as measured by inhibition-of-cytopathic-effect assays. BPR3P0128 appeared to target the viral replication cycle but had no effect on viral adsorption. The inhibition of cap-dependent mRNA transcription by BPR3P0128 was more prominent with a concurrent increase in cap-independent cRNA replication in a primer extension assay, suggesting a role of BPR3P0128 in switching transcription to replication. This reduction in mRNA expression resulted from the BPR3P-mediated inhibition of the cap-dependent endoribonuclease (cap-snatching) activities of nuclear extracts containing the influenza virus polymerase complex. No inhibition of binding of 5' viral RNA to the viral polymerase complex by this compound was detected. BPR3P0128 also effectively inhibited other RNA viruses, such as enterovirus 71 and human rhinovirus, but not DNA viruses, suggesting that BPR3P0128 targets a cellular factor(s) associated with viral PB2 cap-snatching activity. The identification of this factor(s) could help redefine the regulation of viral transcription and replication and thereby provide a potential target for antiviral chemotherapeutics.