RESUMO
Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.
Assuntos
COVID-19 , RNA Viral , Humanos , COVID-19/metabolismo , Endonucleases/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação ViralRESUMO
To obtain a better understanding of the biology behind life-threatening fungal infections caused by Candida albicans, we recently conducted an in silico screening for fungal and host protein interaction partners. We report here that the extracellular domain of human CD4 binds to the moonlighting protein enolase 1 (Eno1) of C. albicans as predicted bioinformatically. By using different anti-CD4 monoclonal antibodies, we determined that C. albicans Eno1 (CaEno1) primarily binds to the extracellular domain 3 of CD4. Functionally, we observed that CaEno1 binding to CD4 activated lymphocyte-specific protein tyrosine kinase (LCK), which was also the case for anti-CD4 monoclonal antibodies tested in parallel. CaEno1 binding to naïve human CD4+ T cells skewed cytokine secretion toward a Th2 profile indicative of poor fungal control. Moreover, CaEno1 inhibited human memory CD4+ T-cell recall responses. Therapeutically, CD4+ T cells transduced with a p41/Crf1-specific T-cell receptor developed for adoptive T-cell therapy were not inhibited by CaEno1 in vitro. Together, the interaction of human CD4+ T cells with CaEno1 modulated host CD4+ T-cell responses in favor of the fungus. Thus, CaEno1 mediates not only immune evasion through its interference with complement regulators but also through the direct modulation of CD4+ T-cell responses.
Assuntos
Candida albicans , Linfócitos T , Humanos , Linfócitos T/metabolismo , Linfócitos T CD4-Positivos , Fosfopiruvato Hidratase/metabolismo , Anticorpos Monoclonais/metabolismoRESUMO
The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAMlow CD103- XCR1- CD172a+ CD11b+ cDC2 from BM by treatment with a combination of Flt3L, LIF, and IL-10 (collectively named as FL10). FL10 promotes common dendritic cell progenitors (CDP) proliferation in the cultures, similar to Flt3L and CDP sorted and cultured in FL10 generate exclusively cDC2. These cDC2 express the transcription factors Irf4, Klf4, and Notch2, and their growth is reduced using BM from Irf4-/- mice, but the expression of Batf3 and Tcf4 is low. Functionally they respond to TLR3, TLR4, and TLR9 signals by upregulation of the surface maturation markers MHC II, CD80, CD86, and CD40, while they poorly secrete proinflammatory cytokines. Peptide presentation to TCR transgenic OT-II cells induced proliferation and IFN-γ production that was similar to GM-CSF-generated BM-DC and higher than Flt3L-generated DC. Together, our data support that FL10 culture of BM cells selectively promotes CDP-derived ESAMlow cDC2 (cDC2B) development and survival in vitro.
Assuntos
Medula Óssea , Interleucina-10 , Animais , Camundongos , Proteína Quinase CDC2 , Moléculas de Adesão CelularRESUMO
Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner.
Assuntos
COVID-19 , Hepatite C Crônica , Humanos , Antivirais/farmacologia , SARS-CoV-2 , Inibidores de Proteases/farmacologia , Indóis/farmacologiaRESUMO
Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Ceramidas/farmacologia , Ceramidas/metabolismo , Replicação Viral , Antivirais/farmacologiaRESUMO
BACKGROUND: Prototype foamy virus (PFV) is nonpathogenic complex retroviruses that express a transcriptional transactivator Tas, which is essential for the activity of viral long terminal repeat (LTR) promoter and internal promoter (IP). Tripartite motif-containing protein 28 (Trim28) is well known as a scaffold protein normally enriched in gene promoter region to repress transcription. We sought to determine if whether Trim28 could be enriched in PFV promoter region to participate the establishment of PFV latency infection. RESULTS: In this study, we show that Trim28 restricts Tas-dependent transactivation activity of PFV promoter and negatively regulates PFV replication. Trim28 was found to be enriched in LTR instead of IP promoter regions of PFV genome and contribute to the maintenance of histone H3K9me3 marks on the LTR promoter. Furthermore, Trim28 interacts with Tas and colocalizes with Tas in the nucleus. Besides, we found that Trim28, an E3 ubiquitin ligase, binds directly to and promotes Tas for ubiquitination and degradation. And the RBCC domain of Trim28 is required for the ubiquitination and degradation of Tas. CONCLUSIONS: Collectively, our findings not only identify a host factor Trim28 negatively inhibits PFV replication by acting as transcriptional restriction factor enriched in viral LTR promoter through modulating H3K9me3 mark here, but also reveal that Trim28 mediated ubiquitin proteasome degradation of Tas as a mechanism underlying Trim28 restricts Tas-dependent transcription activity of PFV promoter and PFV replication. These findings provide new insights into the process of PFV latency establishment.
Assuntos
Histonas/metabolismo , Spumavirus , Proteína 28 com Motivo Tripartido/metabolismo , Linhagem Celular , Humanos , Spumavirus/genética , Sequências Repetidas Terminais , Transativadores/genética , Transativadores/metabolismo , Replicação ViralRESUMO
The main protease of SARS-CoV-2 (Mpro ), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed Mpro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of Mpro and cathepsinâ L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac /Ki =37 500â m-1 s-1 , Ki =24.0â nm) and pyridyl ester 17 (kinac /Ki =29 100â m-1 s-1 , Ki =10.0â nm), promising drug candidates for further development have been discovered.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Nitrilas/farmacologia , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , COVID-19/metabolismo , COVID-19/virologia , Proteases 3C de Coronavírus/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Simulação de Acoplamento Molecular , Nitrilas/química , Inibidores de Proteases/química , Piridinas/química , Piridinas/farmacologia , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Internalização do Vírus/efeitos dos fármacosRESUMO
We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.
Assuntos
Farmacorresistência Viral Múltipla/genética , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Clonagem Molecular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/química , Zidovudina/farmacologiaRESUMO
BACKGROUND: The foamy viral genome encodes four central purine-rich elements localized in the integrase-coding region of pol. Previously, we have shown that the first two of these RNA elements (A and B) are required for protease dimerization and activation. The D element functions as internal polypurine tract during reverse transcription. Peters et al., described the third element (C) as essential for gag expression suggesting that it might serve as an RNA export element for the unspliced genomic transcript. RESULTS: Here, we analysed env splicing and demonstrate that the described C element composed of three GAA repeats known to bind SR proteins regulates env splicing, thus balancing the amount of gag/pol mRNAs. Deletion of the C element effectively promotes a splice site switch from a newly identified env splice acceptor to the intrinsically strong downstream localised env 3' splice acceptor permitting complete splicing of almost all LTR derived transcripts. We provide evidence that repression of this env splice acceptor is a prerequisite for gag expression. This repression is achieved by the C element, resulting in impaired branch point recognition and SF1/mBBP binding. Separating the branch point from the overlapping purine-rich C element, by insertion of only 20 nucleotides, liberated repression and fully restored splicing to the intrinsically strong env 3' splice site. This indicated that the cis-acting element might repress splicing by blocking the recognition of essential splice site signals. CONCLUSIONS: The foamy viral purine-rich C element regulates splicing by suppressing the branch point recognition of the strongest env splice acceptor. It is essential for the formation of unspliced gag and singly spliced pol transcripts.
Assuntos
Regulação Viral da Expressão Gênica , Produtos do Gene gag/genética , Genes env , Genes pol , Purinas/química , Spumavirus/genética , Genoma Viral , Humanos , Splicing de RNA , RNA Viral/genéticaRESUMO
Nucleotide sequences are the fundamental basis for work on molecular mechanisms and for phylogenetic analysis. Recently, we identified sequence errors in all of the LTR sequences of the prototypic foamy virus stored in the GenBank database. Here, we report the resequencing of the proviral plasmids pHSRV13 and pHSRV2. Sequence comparisons revealed an error rate for the foamy virus sequences stored in the database of up to 10 errors per 1000 bp. Even the newest sequences of the codon-optimized foamy virus synthetic Gag, Pol, and Env amino acid sequences showed exchanges compared to the new proviral pHSRV13n sequence. Our results provide evidence that some prototypic foamy virus sequences contain errors and should be revised.
Assuntos
Bases de Dados de Ácidos Nucleicos/normas , Plasmídeos/genética , Análise de Sequência de DNA/normas , Spumavirus/genética , Sequência de Bases , Dados de Sequência Molecular , FilogeniaRESUMO
Biallelic mutations in the untranslated regions (UTRs) of mRNAs are rare causes for monogenetic diseases whose mechanisms remain poorly understood. We investigated a 3'UTR mutation resulting in a complex immunodeficiency syndrome caused by decreased mRNA levels of p14/robld3 by a previously unknown mechanism. Here, we show that the mutation creates a functional 5' splice site (SS) and that its recognition by the spliceosomal component U1 snRNP causes p14 mRNA suppression in the absence of splicing. Histone processing signals are able to rescue p14 expression. Therefore, the mutation interferes only with canonical poly(A)-site 3' end processing. Our data suggest that U1 snRNP inhibits cleavage or poly(A) site recognition. This is the first description of a 3'UTR mutation that creates a functional 5'SS causative of a monogenetic disease. Moreover, our data endorse the recently described role of U1 snRNP in suppression of intronic poly(A) sites, which is here deleterious for p14 mRNA biogenesis.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Síndromes de Imunodeficiência/genética , Neutropenia/congênito , Poliadenilação/genética , Sítios de Splice de RNA/genética , RNA Nuclear Pequeno/genética , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sequência de Bases , Sequência Conservada , Endossomos/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Histonas/fisiologia , Humanos , Íntrons/genética , Mamíferos/genética , Dados de Sequência Molecular , Morfolinos/farmacologia , Neutropenia/genética , Mutação Puntual , Poliadenilação/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Estabilidade de RNA , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Vírus da Dengue/enzimologia , HIV-1/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Replicação Viral/efeitos dos fármacosRESUMO
NS2B-NS3 is a serine protease of the Dengue virus considered a key target in the search for new antiviral drugs. In this study flavonoids were found to be inhibitors of NS2B-NS3 proteases of the Dengue virus serotypes 2 and 3 with IC50 values ranging from 15 to 44 µM. Agathisflavone (1) and myricetin (4) turned out to be noncompetitive inhibitors of dengue virus serotype 2 NS2B-NS3 protease with Ki values of 11 and 4.7 µM, respectively. Docking studies propose a binding mode of the flavonoids in a specific allosteric binding site of the enzyme. Analysis of biomolecular interactions of quercetin (5) with NT647-NHS-labeled Dengue virus serotype 3 NS2B-NS3 protease by microscale thermophoresis experiments, yielded a dissociation constant KD of 20 µM. Our results help to understand the mechanism of inhibition of the Dengue virus serine protease by flavonoids, which is essential for the development of improved inhibitors.
Assuntos
Vírus da Dengue/enzimologia , Flavonoides/farmacologia , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Antivirais/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Flavonoides/química , Cinética , Modelos Moleculares , Simulação de Acoplamento Molecular , Serina Endopeptidases/metabolismo , Relação Estrutura-AtividadeRESUMO
In contrast to orthoretroviruses, processing of foamy viral p71 Gag is limited to a single cleavage site. Nevertheless, Gag maturation is essential for infectivity, but deletion of p3 results in a modest drop in infectivity. Here, we show that Gag processing of p71 to p68 and p3 is essential for full-length cDNA synthesis, while inactivation of Gag cleavage results in cDNAs containing only the RU5 region; cDNAs encompassing the U3 region were almost undetectable.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , DNA Complementar/biossíntese , Produtos do Gene gag/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , DNA Polimerase Dirigida por RNA/metabolismo , Spumavirus/metabolismo , Western Blotting , Produtos do Gene gag/genética , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional/genética , Spumavirus/genética , Spumavirus/patogenicidadeRESUMO
BACKGROUND: Herpesviruses are common animal and human pathogens that cause severe health problems in children, immunocompromised patients, and infected animals with a host range from fish to mammals. Anthocyanin-containing plant extracts have been described as potent antivirals, which might cause fewer harmful side effects than direct-acting antivirals. Here, we report that an extract of Aristotelia chilensis (Molina) Stuntz (Elaeocarpaceae) (MBE) with a high content of the anthocyanin delphinidin suppresses lytic replication of equine, murine and human herpesviruses of replication in vitro. METHODS: We treated cultured cells with MBE and purified individual anthocyanins present in the extract to determine the most active compound at different concentrations. We subsequently infected the cultures with human herpesviruses 1 (HSV-1) or 8 (HHV-8), murine cytomegalovirus (CMV), or equine herpesviruses 1 (EHV-1) and determined the number of infected cells and viral infectivity. RESULTS: MBE inhibited the HSV-1, murine CMV, and EHV-1 by up to 2 orders of magnitude. In the presence of the stabilizing randomly methylated-beta-cyclodextrin, the inhibitory concentration could be lowered significantly. We identified delphinidin as an active antiviral compound and showed that the non-glycosylated delphinidin solved and stabilized with sulfobutylether-beta-cyclodextrin allowed usage of approximately 50 times lower concentrations. CONCLUSION: Glycosylated delphinidin derivatives were identified as active antiviral compounds of MBE. This suggests that plant extracts rich in delphinidin-anthocyanins have potent antiviral properties that could be used in treatment and prevention.
Assuntos
Infecções por Citomegalovirus , Elaeocarpaceae , Hepatite C Crônica , Herpesvirus Humano 1 , Criança , Humanos , Animais , Cavalos , Camundongos , Antocianinas/farmacologia , Antocianinas/análise , Antivirais/farmacologia , Extratos Vegetais/farmacologia , MamíferosRESUMO
Nasal xenobiotic metabolizing enzymes (XMEs) are important for the sense of smell because they influence odorant availability and quality. Since the major part of the human nasal cavity is lined by a respiratory mucosa, we hypothesized that this tissue contributed to nasal odorant metabolism through XME activity. Thus, we built human respiratory tissue models and characterized the XME profiles using single-cell RNA sequencing. We focused on the XMEs dicarbonyl and l-xylulose reductase, aldehyde dehydrogenase (ALDH) 1A1, and ALDH3A1, which play a role in food odorant metabolism. We demonstrated protein abundance and localization in the tissue models and showed the metabolic activity of the corresponding enzyme families by exposing the models to the odorants 3,4-hexandione and benzaldehyde. Using gas chromatography coupled with mass spectrometry, we observed, for example, a significantly higher formation of the corresponding metabolites 4-hydroxy-3-hexanone (39.03 ± 1.5%, p = 0.0022), benzyl alcohol (10.05 ± 0.88%, p = 0.0008), and benzoic acid (8.49 ± 0.57%, p = 0.0004) in odorant-treated tissue models compared to untreated controls (0 ± 0, 0.12 ± 0.12, and 0.18 ± 0.18%, respectively). This is the first study that reveals the XME profile of tissue-engineered human respiratory mucosa models and demonstrates their suitability to study nasal odorant metabolism.
Assuntos
Odorantes , Mucosa Respiratória , Humanos , Odorantes/análise , Mucosa Respiratória/metabolismo , Modelos Biológicos , Cromatografia Gasosa-Espectrometria de Massas , Família Aldeído Desidrogenase 1/metabolismo , Família Aldeído Desidrogenase 1/genética , Xenobióticos/metabolismoRESUMO
BACKGROUND: During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5'LTR and activate polyadenylation in the 3'LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. RESULTS: Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5'LTR. In contrast, polyadenylation at the 3'LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. CONCLUSION: Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5'end of their RNA. At the 3'end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression.
Assuntos
Poli A/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Spumavirus/fisiologia , Animais , Linhagem Celular , Cricetinae , Conformação de Ácido Nucleico , Poliadenilação , Sinais de Poliadenilação na Ponta 3' do RNA , Sítios de Splice de RNA , Sequências Repetidas TerminaisRESUMO
Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.
Assuntos
COVID-19 , Tilorona , Humanos , Fluoxetina , SARS-CoV-2 , Antivirais/farmacologia , Linhagem Celular , CloroquinaRESUMO
By combining docking and molecular dynamics simulations, we explored a library of 65 mostly axially chiral naphthylisoquinoline alkaloids and their analogues, with most different molecular architectures and structural analogues, for their activity against SARS-CoV-2. Although natural biaryls are often regarded without consideration of their axial chirality, they can bind to protein targets in an atroposelective manner. By combining docking results with steered molecular dynamics simulations, we identified one alkaloid, korupensamine A, that atropisomer-specifically inhibited the main protease (Mpro) activity of SARS-CoV-2 significantly in comparison to the reference covalent inhibitor GC376 (IC50 = 2.52 ± 0.14 and 0.88 ± 0.15 µM, respectively) and reduced viral growth by five orders of magnitude in vitro (EC50 = 4.23 ± 1.31 µM). To investigate the binding pathway and mode of interaction of korupensamine A within the active site of the protease, we utilized Gaussian accelerated molecular dynamics simulations, which reproduced the docking pose of korupensamine A inside the active site of the enzyme. The study presents naphthylisoquinoline alkaloids as a new class of potential anti-COVID-19 agents.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Inibidores de Proteases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Hidrolases/metabolismoRESUMO
BACKGROUND: Recently, contradictory results on foamy virus protease activity were published. While our own results indicated that protease activity is regulated by the viral RNA, others suggested that the integrase is involved in the regulation of the protease. RESULTS: To solve this discrepancy we performed additional experiments showing that the protease-reverse transcriptase (PR-RT) exhibits protease activity in vitro and in vivo, which is independent of the integrase domain. In contrast, Pol incorporation, and therefore PR activity in the viral context, is dependent on the integrase domain. To further analyse the regulation of the protease, we incorporated Pol in viruses by expressing a GagPol fusion protein, which supported near wild-type like infectivity. A GagPR-RT fusion, lacking the integrase domain, also resulted in wild-type like Gag processing, indicating that the integrase is dispensable for viral Gag maturation. Furthermore, we demonstrate with a trans-complementation assays that the PR in the context of the PR-RT protein supports in trans both, viral maturation and infectivity. CONCLUSION: We provide evidence that the FV integrase is required for Pol encapsidation and that the FV PR activity is integrase independent. We show that an active PR can be encapsidated in trans as a GagPR-RT fusion protein.