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SARS-CoV-2 is associated with broad tissue tropism, a characteristic often determined by the availability of entry receptors on host cells. Here, we show that TMEM106B, a lysosomal transmembrane protein, can serve as an alternative receptor for SARS-CoV-2 entry into angiotensin-converting enzyme 2 (ACE2)-negative cells. Spike substitution E484D increased TMEM106B binding, thereby enhancing TMEM106B-mediated entry. TMEM106B-specific monoclonal antibodies blocked SARS-CoV-2 infection, demonstrating a role of TMEM106B in viral entry. Using X-ray crystallography, cryogenic electron microscopy (cryo-EM), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we show that the luminal domain (LD) of TMEM106B engages the receptor-binding motif of SARS-CoV-2 spike. Finally, we show that TMEM106B promotes spike-mediated syncytium formation, suggesting a role of TMEM106B in viral fusion. Together, our findings identify an ACE2-independent SARS-CoV-2 infection mechanism that involves cooperative interactions with the receptors heparan sulfate and TMEM106B.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Ligação Proteica , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismoRESUMO
The expanding pandemic of coronavirus disease 2019 (COVID-19) requires the development of safe, efficacious and fast-acting vaccines. Several vaccine platforms are being leveraged for a rapid emergency response1. Here we describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. We assess vaccine safety, immunogenicity and efficacy in several animal models. YF-S0 has an excellent safety profile and induces high levels of SARS-CoV-2 neutralizing antibodies in hamsters (Mesocricetus auratus), mice (Mus musculus) and cynomolgus macaques (Macaca fascicularis), and-concomitantly-protective immunity against yellow fever virus. Humoral immunity is complemented by a cellular immune response with favourable T helper 1 polarization, as profiled in mice. In a hamster model2 and in macaques, YF-S0 prevents infection with SARS-CoV-2. Moreover, a single dose conferred protection from lung disease in most of the vaccinated hamsters within as little as 10 days. Taken together, the quality of the immune responses triggered and the rapid kinetics by which protective immunity can be attained after a single dose warrant further development of this potent SARS-CoV-2 vaccine candidate.
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Vacinas contra COVID-19/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vetores Genéticos/genética , SARS-CoV-2/imunologia , Vacinas Atenuadas/imunologia , Vacina contra Febre Amarela/genética , Animais , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/genética , Cricetinae , Modelos Animais de Doenças , Feminino , Glicosilação , Macaca fascicularis/genética , Macaca fascicularis/imunologia , Macaca fascicularis/virologia , Masculino , Mesocricetus/genética , Mesocricetus/imunologia , Mesocricetus/virologia , Camundongos , Segurança , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genéticaRESUMO
TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.
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Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal , Proteínas de Ligação a DNA/genética , Desacetilase 6 de Histona/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Mutação de Sentido IncorretoRESUMO
Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.
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Esclerose Lateral Amiotrófica/metabolismo , Arginina/metabolismo , Grânulos Citoplasmáticos/metabolismo , Dipeptídeos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Arginina/química , Proteína C9orf72 , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Grânulos Citoplasmáticos/patologia , DNA Helicases , Dipeptídeos/química , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Gotículas Lipídicas/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Domínios Proteicos , Proteínas/química , RNA/metabolismo , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Fatores de Tempo , TransfecçãoRESUMO
Several key functions of the androgen receptor (AR) such as hormone recognition and co-regulator recruitment converge in the ligand binding domain (LBD). Loss- or gain-of-function of the AR contributes to pathologies such as the androgen insensitivity syndrome and prostate cancer. Here, we describe a gain-of-function mutation of the surface-exposed threonine at position 850, located at the amino-terminus of Helix 10 (H10) in the AR LBD. Since T850 phosphorylation was reported to affect AR function, we created the phosphomimetic mutation T850D. The AR T850D variant has a 1.5- to 2-fold increased transcriptional activity with no effect on ligand affinity. In the androgen responsive LNCaP cell line grown in medium with low androgen levels, we observed a growth advantage for cells in which the endogenous AR was replaced by AR T850D. Despite the distance to the AF2 site, the AR T850D LBD displayed an increased affinity for coactivator peptides as well as the 23FQNLF27 motif of AR itself. Molecular Dynamics simulations confirm allosteric transmission of the T850D mutation towards the AF2 site via extended hydrogen bond formation between coactivator peptide and AF2 site. This mechanistic study thus confirms the gain-of-function character of T850D and T850 phosphorylation for AR activity and reveals details of the allosteric communications within the LBD.
Assuntos
Mutação/genética , Receptores Androgênicos/química , Receptores Androgênicos/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Mutação com Ganho de Função/genética , Humanos , Cinética , Ligantes , Lisina/metabolismo , Masculino , Modelos Moleculares , Fosforilação , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , UbiquitinaçãoRESUMO
To curb viral epidemics and pandemics, antiviral drugs are needed with activity against entire genera or families of viruses. Here, we develop a cell-based multiplex antiviral assay for high-throughput screening against multiple viruses at once, as demonstrated by using three distantly related orthoflaviviruses: dengue, Japanese encephalitis and yellow fever virus. Each virus is tagged with a distinct fluorescent protein, enabling individual monitoring in cell culture through high-content imaging. Specific antisera and small-molecule inhibitors are employed to validate that multiplexing approach yields comparable inhibition profiles to single-virus infection assays. To facilitate downstream analysis, a kernel is developed to deconvolute and reduce the multidimensional quantitative data to three cartesian coordinates. The methodology is applicable to viruses from different families as exemplified by co-infections with chikungunya, parainfluenza and Bunyamwera viruses. The multiplex approach is expected to facilitate the discovery of broader-spectrum antivirals, as shown in a pilot screen of approximately 1200 drug-like small-molecules.
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Viroses , Vírus , Humanos , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Técnicas de Cultura de Células , Replicação ViralRESUMO
Ebola virus (EBOV) and related filoviruses such as Sudan virus (SUDV) threaten global public health. Effective filovirus vaccines are available only for EBOV, yet restricted to emergency use considering a high reactogenicity and demanding logistics. Here we present YF-EBO, a live YF17D-vectored dual-target vaccine candidate expressing EBOV glycoprotein (GP) as protective antigen. Safety of YF-EBO in mice was further improved over that of parental YF17D vaccine. A single dose of YF-EBO was sufficient to induce high levels of EBOV GP-specific antibodies and cellular immune responses, that protected against lethal infection using EBOV GP-pseudotyped recombinant vesicular stomatitis virus (rVSV-EBOV) in interferon-deficient (Ifnar-/-) mice as surrogate challenge model. Concomitantly induced yellow fever virus (YFV)-specific immunity protected Ifnar-/- mice against intracranial YFV challenge. YF-EBO could thus help to simultaneously combat both EBOV and YFV epidemics. Finally, we demonstrate how to target other highly pathogenic filoviruses such as SUDV at the root of the 2022 outbreak in Uganda.
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BACKGROUND: Sexual acquisition of the human immunodeficiency virus (HIV) through mucosal transmission may be prevented by using topically applied agents that block HIV transmission from one individual to another. Therefore, virucidal agents that inactivate HIV virions may be used as a component in topical microbicides. RESULTS: Here, we have identified 2-methyl-3-phenyl-2H-[1,2,4]thiadiazol-5-ylideneamine (WDO-217) as a low-molecular-weight molecule that inactivates HIV particles. Both HIV-1 and HIV-2 virions pretreated with this compound were unable to infect permissive cells. Moreover, WDO-217 was able to inhibit infections of a wide spectrum of wild-type and drug-resistant HIV-1, including clinical isolates, HIV-2 and SIV strains. Whereas the capture of virus by DC-SIGN was unaffected by the compound, it efficiently prevented the transmission of DC-SIGN-captured virus to CD4+ T-lymphocytes. Interestingly, exposure of virions to WDO-217 reduced the amount of virion-associated genomic RNA as measured by real-time RT-qPCR. Further mechanism-of-action studies demonstrated that WDO-217 efficiently ejects zinc from the zinc fingers of the retroviral nucleocapsid protein NCp7 and inhibits the cTAR destabilization properties of this protein. Importantly, WDO-217 was able to eject zinc from both zinc fingers, even when NCp7 was bound to oligonucleotides, while no covalent interaction between NCp7 and WDO-217 could be observed. CONCLUSION: This compound is a new lead structure that can be used for the development of a new series of NCp7 zinc ejectors as candidate topical microbicide agents.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Nucleocapsídeo/metabolismo , Tiadiazóis/farmacologia , Vírion/efeitos dos fármacos , Inativação de Vírus/efeitos dos fármacos , Zinco/metabolismo , Fármacos Anti-HIV/química , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , HIV-1/química , HIV-1/fisiologia , HIV-2/química , HIV-2/fisiologia , Humanos , Nucleocapsídeo/química , Tiadiazóis/química , Vírion/química , Vírion/fisiologia , Dedos de ZincoRESUMO
Stress granules are non-membrane bound granules temporarily forming in the cytoplasm in response to stress. Proteins of the nucleocytoplasmic transport machinery were found in these stress granules and it was suggested that stress granules contribute to the nucleocytoplasmic transport defects in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The aim of this study was to investigate whether there is a causal link between stress granule formation and nucleocytoplasmic transport deficits. Therefore, we uncoupled stress granule formation from cellular stress while studying nuclear import. This was carried out by preventing cells from assembling stress granules despite being subjected to cellular stress either by knocking down both G3BP1 and G3BP2 or by pharmacologically inhibiting stress granule formation. Conversely, we induced stress granules by overexpressing G3BP1 in the absence of cellular stress. In both conditions, nuclear import was not affected demonstrating that stress granule formation is not a direct cause of stress-induced nucleocytoplasmic transport deficits.
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To control the coronavirus disease 2019 (COVID-19) pandemic and the emergence of different variants of concern (VoCs), novel vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed. In this study, we report the potent immunogenicity and efficacy induced in hamsters by a vaccine candidate based on a modified vaccinia virus Ankara (MVA) vector expressing a human codon optimized full-length SARS-CoV-2 spike (S) protein (MVA-S). Immunization with one or two doses of MVA-S elicited high titers of S- and receptor-binding domain (RBD)-binding IgG antibodies and neutralizing antibodies against parental SARS-CoV-2 and VoC alpha, beta, gamma, delta, and omicron. After SARS-CoV-2 challenge, MVA-S-vaccinated hamsters showed a significantly strong reduction of viral RNA and infectious virus in the lungs compared to the MVA-WT control group. Moreover, a marked reduction in lung histopathology was also observed in MVA-S-vaccinated hamsters. These results favor the use of MVA-S as a potential vaccine candidate for SARS-CoV-2 in clinical trials.
Assuntos
COVID-19 , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Cricetinae , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Vaccinia virus/genéticaRESUMO
Current COVID-19 vaccines are based on prototypic spike sequences from ancestral 2019 SARS-CoV-2 strains. However, the ongoing pandemic is fueled by variants of concern (VOC) escaping vaccine-mediated protection. Here we demonstrate how immunization in hamsters using prototypic spike expressed from yellow fever 17D (YF17D) as vector blocks ancestral virus (B lineage) and VOC Alpha (B.1.1.7) yet fails to fully protect from Beta (B.1.351). However, the same YF17D vectored vaccine candidate with an evolved antigen induced considerably improved neutralizing antibody responses against VOCs Beta, Gamma (P.1) and the recently predominant Omicron (B.1.1.529), while maintaining immunogenicity against ancestral virus and VOC Delta (B.1.617.2). Thus vaccinated animals resisted challenge by all VOCs, including vigorous high titre exposure to the most difficult to cover Beta, Delta and Omicron variants, eliminating detectable virus and markedly improving lung pathology. Finally, vaccinated hamsters did not transmit Delta variant to non-vaccinated cage mates. Overall, our data illustrate how current first-generation COVID-19 vaccines may need to be updated to maintain efficacy against emerging VOCs and their spread at community level.
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COVID-19 , Vacinas Virais , Vacina contra Febre Amarela , Cricetinae , Animais , Humanos , SARS-CoV-2/genética , Vacinas Virais/genética , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
BACKGROUND: The live-attenuated yellow fever vaccine YF17D holds great promise as alternative viral vector vaccine platform, showcased by our previously presented potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate YF-S0. Besides protection from SARS-CoV-2, YF-S0 also induced strong yellow fever virus (YFV)-specific immunity, suggestive for full dual activity. A vaccine concomitantly protecting from SARS-CoV-2 and YFV would be of great benefit for those living in YFV-endemic areas with limited access to current SARS-CoV-2 vaccines. However, for broader applicability, pre-existing vector immunity should not impact the potency of such YF17D-vectored vaccines. METHODS: The immunogenicity and efficacy of YF-S0 against YFV and SARS-CoV-2 in the presence of strong pre-existing YFV immunity were evaluated in mouse and hamster challenge models. FINDINGS: Here, we show that a single dose of YF-S0 is sufficient to induce strong humoral and cellular immunity against YFV as well as SARS-CoV-2 in mice and hamsters; resulting in full protection from vigorous YFV challenge in either model; in mice against lethal intracranial YF17D challenge, and in hamsters against viscerotropic infection and liver disease following challenge with highly pathogenic hamster-adapted YFV-Asibi strain. Importantly, strong pre-existing immunity against the YF17D vector did not interfere with subsequent YF-S0 vaccination in mice or hamsters; nor with protection conferred against SARS-CoV-2 strain B1.1.7 (Alpha variant) infection in hamsters. INTERPRETATION: Our findings warrant the development of YF-S0 as dual SARS-CoV-2 and YFV vaccine. Contrary to other viral vaccine platforms, use of YF17D does not suffer from pre-existing vector immunity. FUNDING: Stated in the acknowledgments.
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COVID-19 , Vacinas Virais , Vacina contra Febre Amarela , Febre Amarela , Animais , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Cricetinae , Humanos , Camundongos , SARS-CoV-2 , Vacinas Virais/genética , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/genéticaRESUMO
To mitigate the massive COVID-19 burden caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccination campaigns were initiated. We performed a single-center observational trial to monitor the mid- (3 months) and long-term (10 months) adaptive immune response and to document breakthrough infections (BTI) in healthcare workers (n = 84) upon BNT162b2 vaccination in a real-world setting. Firstly, serology was determined through immunoassays. Secondly, antibody functionality was analyzed via in vitro binding inhibition and pseudovirus neutralization and circulating receptor-binding domain (RBD)-specific B cells were assessed. Moreover, the induction of SARS-CoV-2-specific T cells was investigated by an interferon-γ release assay combined with flowcytometric profiling of activated CD4+ and CD8+ T cells. Within individuals that did not experience BTI (n = 62), vaccine-induced humoral and cellular immune responses were not correlated. Interestingly, waning over time was more pronounced within humoral compared to cellular immunity. In particular, 45 of these 62 subjects no longer displayed functional neutralization against the delta variant of concern (VoC) at long-term follow-up. Noteworthily, we reported a high incidence of symptomatic BTI cases (17.11%) caused by alpha and delta VoCs, although vaccine-induced immunity was only slightly reduced compared to subjects without BTI at mid-term follow-up.
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COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , Bélgica , Linfócitos T CD8-Positivos , COVID-19/epidemiologia , COVID-19/prevenção & controle , Progressão da Doença , Seguimentos , Pessoal de Saúde , Humanos , Imunidade Celular , Imunidade Humoral , Incidência , SARS-CoV-2/genética , VacinaçãoRESUMO
Treatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent patient with COVID-19, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 D614G, beta, gamma, and delta infection in vitro, with three mAbs neutralizing omicron as well. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta, and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 µg/mL, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.
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Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics.
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Encefalite Japonesa , Febre Amarela , Infecção por Zika virus , Zika virus , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Testes de Neutralização/métodos , Febre Amarela/diagnóstico , Febre Amarela/epidemiologia , Febre Amarela/prevenção & controle , Vírus da Febre AmarelaRESUMO
The human immunodeficiency virus, type 1 (HIV-1)-encoded Rev protein is essential for the expression of late viral mRNAs. Rev forms a large organized multimeric protein-protein complex on the Rev response element of these viral mRNA species and transports them from the nucleus to the cytoplasm, exploiting the CRM1-mediated cellular machinery. Here we report the selection of a nanobody, derived from a llama heavy-chain only antibody, that efficiently blocks the assembly of Rev multimers. The nanobody inhibits HIV-1 replication in cells and specifically suppresses the Rev-dependent expression of partially spliced and unspliced HIV-1 RNA. In HIV-susceptible cells, this nanobody thus has potential as an effective anti-HIV agent using genetic immunization strategies. Its binding site was mapped to Rev residues Lys-20 and Tyr-23 located in the N-terminal alpha-helical multimerization domain. In the presence of this nanobody, we observed an accumulation of dimeric Rev species, supporting a head-to-head/tail-to-tail molecular model for Rev assembly. The results indicate that the oligomeric assembly of Rev follows an ordered stepwise process and identify a new epitope within Rev that could guide strategies for the development of novel HIV inhibitors.
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Produtos do Gene rev/química , Infecções por HIV/tratamento farmacológico , HIV-1/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/farmacologia , Anticorpos/química , Camelídeos Americanos , Dimerização , Transferência Ressonante de Energia de Fluorescência , Produtos do Gene rev/metabolismo , Células HeLa , Humanos , Lisina/química , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tirosina/químicaRESUMO
The export of viral RNA from the nucleus to the cytoplasm of the cellular host is a crucial step in the life cycle of HIV-1 that is mediated by the viral Rev protein. One aspect of the Rev function, its multimerization, is still unexplored as a target for antiviral therapy. This is partly due to the lack of a fast and solid system to measure Rev multimerization. We have developed a high throughput in vitro Rev multimerization assay based on fluorescence resonance energy transfer (FRET) in which real-time Rev-Rev interactions can be measured both in the absence and the presence of Rev specific RRE RNA. Well-characterized Rev multimerization deficient mutants showed reduced FRET as well as unlabeled Rev molecules were able to inhibit the FRET signal demonstrating the specificity of the assay. Upon multimerization along RRE RNA the FRET signal significantly increased but dropped again at equimolar Rev/RRE ratios suggesting that in this condition most Rev molecules are bound as monomers to the RRE. Furthermore, using this assay, we demonstrate that a previously selected llama heavy-chain only antibody was shown to not only prevent the development of Rev multimers but also disassemble the already formed complexes confirming the dynamic nature of the Rev-Rev interactions. The in vitro FRET based multimerization assay facilitates the further study of the basic mechanism of cooperative Rev multimerization along the RRE and is also widely applicable to study the assembly of other functional complexes involving protein homo-multimerization or cooperative protein-protein interactions on RNA or DNA.
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Transferência Ressonante de Energia de Fluorescência/métodos , Produtos do Gene rev/metabolismo , HIV-1/metabolismo , Multimerização Proteica , Produtos do Gene rev/genética , Genes env/genética , HIV-1/genética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte de RNA , RNA Viral/genéticaRESUMO
The live-attenuated yellow fever 17D (YF17D) vaccine is one of the most efficacious human vaccines and also employed as a vector for novel vaccines. However, in the lack of appropriate immunocompetent small animal models, mechanistic insight in YF17D-induced protective immunity remains limited. To better understand YF17D vaccination and to identify a suitable mouse model, we evaluated the immunogenicity and protective efficacy of YF17D in five complementary mouse models, i.e. wild-type (WT) BALB/c, C57BL/6, IFN-α/ß receptor (IFNAR-/-) deficient mice, and in WT mice in which type I IFN signalling was temporally ablated by an IFNAR blocking (MAR-1) antibody. Alike in IFNAR-/- mice, YF17D induced in either WT mice strong humoral immune responses dominated by IgG2a/c isotype (Th1 type) antibodies, yet only when IFNAR was blocked. Vigorous cellular immunity characterized by CD4+ T-cells producing IFN-γ and TNF-α were mounted in MAR-1 treated C57BL/6 and in IFNAR-/- mice. Surprisingly, vaccine-induced protection was largely mouse model dependent. Full protection against lethal intracranial challenge and a massive reduction of virus loads was conferred already by a minimal dose of 2 PFU YF17D in BALB/c and IFNAR-/- mice, but not in C57BL/6 mice. Correlation analysis of infection outcome with pre-challenge immunological markers indicates that YFV-specific IgG might suffice for protection, even in the absence of detectable levels of neutralizing antibodies. Finally, we propose that, in addition to IFNAR-/- mice, C57BL/6 mice with temporally blocked IFN-α/ß receptors represent a promising immunocompetent mouse model for the study of YF17D-induced immunity and evaluation of YF17D-derived vaccines.
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Vacina contra Febre Amarela/administração & dosagem , Vacina contra Febre Amarela/imunologia , Febre Amarela/prevenção & controle , Vírus da Febre Amarela/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Febre Amarela/imunologia , Febre Amarela/virologia , Vacina contra Febre Amarela/genética , Vírus da Febre Amarela/genéticaRESUMO
Effective agents to treat coronavirus infection are urgently required, not only to treat COVID-19, but to prepare for future outbreaks. Repurposed anti-virals such as remdesivir and human anti-inflammatories such as barcitinib have received emergency approval but their overall benefits remain unclear. Vaccines are the most promising prospect for COVID-19, but will need to be redeveloped for any future coronavirus outbreak. Protecting against future outbreaks requires the identification of targets that are conserved between coronavirus strains and amenable to drug discovery. Two such targets are the main protease (Mpro) and the papain-like protease (PLpro) which are essential for the coronavirus replication cycle. We describe the discovery of two non-antiviral therapeutic agents, the caspase-1 inhibitor SDZ 224015 and Tarloxotinib that target Mpro and PLpro, respectively. These were identified through extensive experimental screens of the drug repurposing ReFRAME library of 12,000 therapeutic agents. The caspase-1 inhibitor SDZ 224015, was found to be a potent irreversible inhibitor of Mpro (IC50 30 nM) while Tarloxotinib, a clinical stage epidermal growth factor receptor inhibitor, is a sub micromolar inhibitor of PLpro (IC50 300 nM, Ki 200 nM) and is the first reported PLpro inhibitor with drug-like properties. SDZ 224015 and Tarloxotinib have both undergone safety evaluation in humans and hence are candidates for COVID-19 clinical evaluation.
Assuntos
Antivirais/química , Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Reposicionamento de Medicamentos , Oligopeptídeos/química , Linhagem Celular , Humanos , Serpinas/química , Proteínas Virais/químicaRESUMO
Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these experiments can be performed without the need for expensive equipment and have the potential to be upscaled for high-throughput screening purposes.