RESUMO
HIV-1 successfully establishes long-term infection in its target cells despite viral cytotoxic effects. We have recently shown that cell metabolism is an important factor driving CD4+ T cell susceptibility to HIV-1 and the survival of infected cells. We show here that expression of antiapoptotic clone 11 (AAC-11), an antiapoptotic factor upregulated in many cancers, increased with progressive CD4+ T cell memory differentiation in association with the expression of cell cycle, activation, and metabolism genes and was correlated with susceptibility to HIV-1 infection. Synthetic peptides based on the LZ domain sequence of AAC-11, responsible for its interaction with molecular partners, were previously shown to be cytotoxic to cancer cells. Here, we observed that these peptides also blocked HIV-1 infection by inducing the death of HIV-1-susceptible primary CD4+ T cells across all T cell subsets. The peptides targeted metabolically active cells and had the greatest effect on effector and transitional CD4+ T cell memory subsets. Our results suggest that the AAC-11 survival pathway is potentially involved in the survival of HIV-1-infectible cells and provide proof of principle that some cellular characteristics can be targeted to eliminate the cells offering the best conditions to sustain HIV-1 replication.IMPORTANCE Although antiretroviral treatment efficiently blocks HIV multiplication, it cannot eliminate cells already carrying integrated proviruses. In the search for an HIV cure, the identification of new potential targets to selectively eliminate infected cells is of the outmost importance. We show here that peptides derived from antiapoptotic clone 11 (AAC-11), whose expression levels correlated with susceptibility to HIV-1 infection of CD4+ T cells, induced cytotoxicity in CD4+ T cells showing the highest levels of activation and metabolic activity, conditions known to favor HIV-1 infection. Accordingly, CD4+ T cells that survived the cytotoxic action of the AAC-11 peptides were resistant to HIV-1 replication. Our results identify a new potential molecular pathway to target HIV-1 infection.
Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Memória Imunológica/efeitos dos fármacos , Proteínas Nucleares/farmacologia , Peptídeos/farmacologia , Replicação Viral/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Suscetibilidade a Doenças , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Humanos , Proteínas Nucleares/química , Peptídeos/química , Domínios Proteicos , Replicação Viral/imunologiaRESUMO
Interferon (IFN) plays a central role in regulating host immune response to viral pathogens through the induction of IFN-Stimulated Genes (ISGs). IFN also enhances cellular SUMOylation and ISGylation, though the functional interplay between these modifications remains unclear. Here, we used a system-level approach to profile global changes in protein abundance in SUMO3-expressing cells stimulated by IFNα. These analyses revealed the stabilization of several ISG factors including SAMHD1, MxB, GBP1, GBP5, Tetherin/BST2 and members of IFITM, IFIT and IFI families. This process was correlated with enhanced IFNα-induced anti-HIV-1 and HSV-1 activities. Also IFNα upregulated protein ISGylation through increased abundance of E2 conjugating enzyme UBE2L6, and E3 ISG15 ligases TRIM25 and HERC5. Remarkably, TRIM25 depletion blocked SUMO3-dependent protein stabilization in response to IFNα. Our data identify a new mechanism by which SUMO3 regulates ISG product stability and reinforces the relevance of the SUMO pathway in controlling both the expression and functions of the restriction factors and IFN antiviral response.
Assuntos
Interferon-alfa/farmacologia , Sumoilação/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismoRESUMO
HIV-1 infection of noncycling cells, such as dendritic cells (DCs), is impaired due to limited availability of deoxynucleoside triphosphates (dNTPs), which are needed for HIV-1 reverse transcription. The levels of dNTPs are tightly regulated during the cell cycle and depend on the balance between dNTP biosynthesis and degradation. SAMHD1 potently blocks HIV-1 replication in DCs, although the underlying mechanism is still unclear. SAMHD1 has been reported to be able to degrade dNTPs and viral nucleic acids, which may both hamper HIV-1 reverse transcription. The relative contribution of these activities may differ in cycling and noncycling cells. Here, we show that inhibition of HIV-1 replication in monocyte-derived DCs (MDDCs) is associated with an increased expression of p21cip1/waf, a cell cycle regulator that is involved in the differentiation and maturation of DCs. Induction of p21 in MDDCs decreases the pool of dNTPs and increases the antiviral active isoform of SAMHD1. Although both processes are complementary in inhibiting HIV-1 replication, the antiviral activity of SAMHD1 in our primary cell model appears to be, at least partially, independent of its dNTPase activity. The reduction in the pool of dNTPs in MDDCs appears rather mostly due to a p21-mediated suppression of several enzymes involved in dNTP synthesis (i.e., RNR2, TYMS, and TK-1). These results are important to better understand the interplay between HIV-1 and DCs and may inform the design of new therapeutic approaches to decrease viral dissemination and improve immune responses against HIV-1.IMPORTANCE DCs play a key role in the induction of immune responses against HIV. However, HIV has evolved ways to exploit these cells, facilitating immune evasion and virus dissemination. We have found that the expression of p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation and monocyte differentiation and maturation, potentially can contribute to the inhibition of HIV-1 replication in monocyte-derived DCs through multiple mechanisms. p21 decreased the size of the intracellular dNTP pool. In parallel, p21 prevented SAMHD1 phosphorylation and promoted SAMHD1 dNTPase-independent antiviral activity. Thus, induction of p21 resulted in conditions that allowed the effective inhibition of HIV-1 replication through complementary mechanisms. Overall, p21 appears to be a key regulator of HIV infection in myeloid cells.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Dendríticas/virologia , Desoxirribonucleotídeos/biossíntese , HIV-1/fisiologia , Monócitos/virologia , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Antivirais/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Replicação do DNA , Células Dendríticas/fisiologia , Desoxirribonucleotídeos/química , HIV-1/imunologia , Humanos , Polifosfatos/química , Polifosfatos/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação ViralRESUMO
The era of antiretroviral therapy has made HIV-1 infection a manageable chronic disease for those with access to treatment. Despite treatment, virus persists in tissue reservoirs seeded with long-lived infected cells that are resistant to cell death and immune recognition. Which cells contribute to this reservoir and which factors determine their persistence are central questions that need to be answered to achieve viral eradication. In this chapter, we describe how cell susceptibility to infection, resistance to cell death, and immune-mediated killing as well as natural cell life span and turnover potential are central components that allow persistence of different lymphoid and myeloid cell subsets that were recently identified as key players in harboring latent and actively replicating virus. The relative contribution of these subsets to persistence of viral reservoir is described, and the open questions are highlighted.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Viremia/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Apoptose , Farmacorresistência Viral , HIV-1/classificação , HIV-1/fisiologia , Humanos , Evasão da Resposta Imune , Células Mieloides/virologia , Receptores de HIV/metabolismo , Subpopulações de Linfócitos T/virologia , Carga Viral , Latência Viral , Replicação ViralRESUMO
This study assessed the anthelmintic resistance in strongylid nematodes against commonly used anthelmintic (AH) drugs in a French galloping racehorse stud farm from March to December 2023. Faecal egg count reduction tests (FECRTs) were conducted in three different groups of Thoroughbred yearlings (a group of 6 males, a group of 13 females and a group of 8 females and 3 males) following the new World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. The efficacy of fenbendazole was tested in two groups once during the monitoring period (in March), the efficacy of ivermectin in 3 groups twice (in March-April and in November-December) and the efficacy of pyrantel in one group once (in May-June). For each FECRT, the 90% confidence interval of the percentage faecal egg count reduction was calculated using the hybrid Frequentist/Bayesian analysis method. The resistance in strongylids was observed to fenbendazole, pyrantel and ivermectin in all the groups in which these drugs were tested. The number of animals in each group was sufficient to reach ≥80% power for the resistance test. The results highlight the first case of triple AH resistance in strongylids in France. Further studies involving more farms and equids are required to assess the prevalence of AH resistance in France and refine recommendations for owners.
Assuntos
Anti-Helmínticos , Doenças dos Cavalos , Animais , Feminino , Masculino , Anti-Helmínticos/farmacologia , Teorema de Bayes , Resistência a Medicamentos , Fazendas , Fezes/parasitologia , Fenbendazol/farmacologia , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/epidemiologia , Doenças dos Cavalos/parasitologia , Cavalos , Ivermectina/farmacologia , Contagem de Ovos de Parasitas/veterinária , Pirantel/farmacologiaRESUMO
Grazing equids are constantly exposed to three clinically important gastrointestinal parasites (small strongyles/cyathostomins, Anoplocephala spp. and Parascaris spp.). Knowledge of the local seasonal dynamic of these parasitic infections is important for constructing a sustainable parasite control program with a rational number of anthelmintic treatments. However, studies describing these patterns are sparse in France. In this context, a two-year study was carried out to assess i) the seasonal dynamic and variability of strongyle faecal egg counts (FEC) and infective larvae (L3) counts on pastures, and ii) the prevalence of Anoplocephala spp. and Parascaris spp. and the dynamic evolution of their presence. During 2021 and 2022 grazing seasons, monthly individual faecal egg counts (FEC) and diarrhea scores (DS) were determined on 428 equids divided into 33 groups. A monthly body condition score (BCS) was also attributed to animals ≥3 years old and a monthly bodyweight was estimated for each animal <3 years old. At the group level, the strongyle L3 counts on grazed pastures were carried out at least in spring, summer and autumn. Eggs of strongyles were observed in 97% of equids. In 64% of the groups, the peaks of FEC were noted in September and October. At the individual level, the maximum strongyle FEC was related to age, group of breeds, number of grazed plots and number of anthelmintic treatments. No negative association was observed between strongyle FEC and BCS or average daily weight gain. In the pastures, cyathostomin larvae were found almost exclusively. Over the two years, the peaks of cyathostomin L3 counts occurred in 87% of the groups between September and November and ranged from 635 to 87,500 L3 kg-1 dry herbage. The variability of the maximum cyathostomin L3 count in each group was explained by the year and the number of grazed plots. Eggs of Anoplocephala spp. were observed in 12% of equids. Eggs of Parascaris spp. were noted in 34% of one year-old animals, 9% of two years-olds and 2% of olders. Anoplocephala spp. and Parascaris spp. eggs were observed every month with a peak in the percentage of shedders in groups in October for Anoplocephala spp. and May-June for Parascaris spp.This study highlights the prevalence of each parasite, the variability in cyathostomin egg excretion and L3 counts amongst groups and individuals and the factors involved in this variation These local epidemiological data will help us to re-think a newer strategy against these parasites.
Assuntos
Anti-Helmínticos , Ascaridoidea , Doenças dos Cavalos , Enteropatias Parasitárias , Parasitos , Humanos , Cavalos , Animais , Doenças dos Cavalos/parasitologia , Estações do Ano , Prevalência , Contagem de Ovos de Parasitas/veterinária , Enteropatias Parasitárias/epidemiologia , Enteropatias Parasitárias/veterinária , Anti-Helmínticos/uso terapêutico , Fezes/parasitologia , França/epidemiologiaRESUMO
Domestic species, including equids, were introduced in the Galapagos Islands in the XIX century. Equine vector-borne diseases are circulating in South America but their occurrence in the Galapagos Island was unknown. The objective of this study was to detect the occurrence of West Nile virus (WNV), Usutu virus (USUV) and equine infectious anemia virus (EIAV) in the four Galapagos Islands raising equids if they were present at a prevalence >1%. Serum samples were collected from 411 equids belonging to 124 owners from April to July 2019. All the results were negative to the ELISA tests used suggesting that WNV, USUV and EIAV are not circulating in the equine population of the Galapagos Islands.
RESUMO
We have performed an equine influenza (EI) serological study of the equine population in Algeria by testing 298 serum samples collected between February and August 2021 from 5 provinces. The results were obtained performing an NP-ELISA. Our results revealed that 49.3% (147/298) samples positive for antibodies to EI (H3N8). During this study and after a gap of one decade an outbreak of EI was reported in Algeria in the first week of March 2021. The disease was confirmed by virus detection from the nasal swabs (n = 39) by qRT-PCR and by identifying 5 EI seroconversion. The virus sequences were identified as H3N8 by sequencing the haemagglutinin (HA) and neuraminidase (NA) genes. Alignment of HA1 amino acid sequence confirmed that viruses belong to Clade 1 of the Florida sublineage in the American lineage. This study indicate the first detection of FC1 strain of EIV in Maghreb area.
Assuntos
Doenças dos Cavalos , Vírus da Influenza A Subtipo H3N8 , Influenza Humana , Infecções por Orthomyxoviridae , Cavalos , Animais , Humanos , Vírus da Influenza A Subtipo H3N8/genética , Argélia/epidemiologia , Influenza Humana/epidemiologia , Filogenia , África do Norte , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Surtos de Doenças/veterinária , Doenças dos Cavalos/diagnósticoRESUMO
BACKGROUND: Expression of the cellular karyopherin TNPO3/transportin-SR2/Tnp3 is necessary for HIV-1 infection. Depletion of TNPO3 expression in mammalian cells inhibits HIV-1 infection after reverse transcription but prior to integration. RESULTS: This work explores the role of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) in the ability of TNPO3-depleted cells to inhibit HIV-1 infection. Our findings showed that depletion of TNPO3 expression inhibits HIV-1 infection, while the simultaneous depletion of TNPO3 and CPSF6 expression rescues HIV-1 infection. Several experiments to understand the rescue of infectivity by CPSF6 were performed. Our experiments revealed that the HIV-1 capsid binding ability of the endogenously expressed CPSF6 from TNPO3-depleted cells does not change when compared to CPSF6 from wild type cells. In agreement with our previous results, depletion of TNPO3 did not change the nuclear localization of CPSF6. Studies on the formation of 2-LRT circles during HIV-1 infection revealed that TNPO3-depleted cells are impaired in the integration process or exhibit a defect in the formation of 2-LTR circles. To understand whether the cytosolic fraction of CPSF6 is responsible for the inhibition of HIV-1 in TNPO3-depleted cells, we tested the ability of a cytosolic full-length CPSF6 to block HIV-1 infection. These results demonstrated that overexpression of a cytosolic full-length CPSF6 blocks HIV-1 infection at the nuclear import step. Fate of the capsid assays revealed that cytosolic expression of CPSF6 enhances stability of the HIV-1 core during infection. CONCLUSIONS: These results suggested that inhibition of HIV-1 by TNPO3-depleted cells requires CPSF6.
Assuntos
HIV-1/imunologia , HIV-1/fisiologia , Transcrição Reversa , Integração Viral , beta Carioferinas/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Linhagem Celular , HumanosRESUMO
BACKGROUND: SAMHD1 is a restriction factor that potently blocks infection by HIV-1 and other retroviruses. We have previously demonstrated that SAMHD1 oligomerizes in mammalian cells by immunoprecipitation. Here we investigated the contribution of SAMHD1 oligomerization to retroviral restriction. RESULTS: Structural analysis of SAMHD1 and homologous HD domain proteins revealed that key hydrophobic residues Y146, Y154, L428 and Y432 stabilize the extensive dimer interface observed in the SAMHD1 crystal structure. Full-length SAMHD1 variants Y146S/Y154S and L428S/Y432S lost their ability to oligomerize tested by immunoprecipitation in mammalian cells. In agreement with these observations, the Y146S/Y154S variant of a bacterial construct expressing the HD domain of human SAMHD1 (residues 109-626) disrupted the dGTP-dependent tetramerization of SAMHD1 in vitro. Tetramerization-defective variants of the full-length SAMHD1 immunoprecipitated from mammalian cells and of the bacterially-expressed HD domain construct lost their dNTPase activity. The nuclease activity of the HD domain construct was not perturbed by the Y146S/Y154S mutations. Remarkably, oligomerization-deficient SAMHD1 variants potently restricted HIV-1 infection. CONCLUSIONS: These results suggested that SAMHD1 oligomerization is not required for the ability of the protein to block HIV-1 infection.
Assuntos
HIV-1/imunologia , Interações Hospedeiro-Patógeno , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Multimerização Proteica , Linhagem Celular , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Imunoprecipitação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HDRESUMO
TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import.
Assuntos
Núcleo Celular/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Integração Viral , Replicação Viral , beta Carioferinas/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virologia , Infecções por HIV/genética , HIV-1/genética , Humanos , Ligação Proteica , beta Carioferinas/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
In order to determine the prevalence of equine infectious anemia virus (EIAV), Usutu virus (USUV), and West Nile virus (WNV) in eastern Algerian drylands, 340 sera from distinct equids have been collected from 2015 to 2017. Serological analysis for the presence of antibodies against EIAV and flaviviruses was performed using commercially available ELISAs. Sera detected positive, doubtful, or negative close to the doubtful threshold in flavivirus ELISA were tested by the virus neutralization test (VNT), using WNV and USUV strains. The prevalence of WNV antibodies with ELISA was 11.47% (39/340) against 13.53% (46/340) by WNV VNT. EIAV antibodies were not detected in any samples. WNV seroprevalence varies with species, breed and location of horses. Only, one equid was positive for both WNV and USUV neutralizing antibodies. This is the first screening on equids sera of EIAV and USUV in Algeria. This study indicate that WNV and possibly USUV have circulated/are circulating in the Algerian equine population, unlike EIAV does not seem to be present.
Assuntos
Infecções por Flavivirus , Flavivirus , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Cavalos , Febre do Nilo Ocidental/veterinária , Infecções por Flavivirus/epidemiologia , Infecções por Flavivirus/veterinária , Estudos Soroepidemiológicos , Anticorpos Antivirais , Fatores de RiscoRESUMO
In diverse brain pathologies, astrocytes become reactive and undergo profound phenotypic changes. Connexin43 (Cx43), the main gap junction channel-forming protein in astrocytes, is one of the proteins modified in reactive astrocytes. Downregulation of Cx43 in cultured astrocytes activates c-Src, promotes proliferation, and increases the rate of glucose uptake; however, so far there have been no studies examining whether this cascade of events takes place in reactive astrocytes. In this work, we analyzed this pathway after a cortical lesion induced by a kainic acid injection. As previously described, astrocytes reacted to the lesion with an increase in glial fibrillary acidic protein and a decrease in Cx43 expression. Some of these reactive astrocytes proliferated, as estimated by bromodeoxyuridine incorporation and cyclins D1 and D3 upregulation. In addition, the expression of the glucose transporter GLUT-3 and the enzyme responsible for glucose phosphorylation, Type II hexokinase (Hx-2), were induced in reactive astrocytes, suggesting an increased glucose uptake. Previous in vitro studies reported that c-Src is the link between Cx43 and glucose uptake and proliferation in astrocytes. Here, we found that c-Src activity increased in the lesioned area. c-Src activation and Cx43 downregulation preceded the peak of Hx-2 and cyclin D3 expression, suggesting that c-Src could mediate the effect of Cx43 on glucose uptake and proliferation in reactive astrocytes after an excitotoxic insult. Interestingly, we identify c-Src, GLUT-3, and Hx-2 in the signaling mechanisms involved in the reaction of astroglia to injury. Altogether these data contribute to identify new therapeutical targets to enhance astrocyte neuroprotective activities.
Assuntos
Astrócitos/metabolismo , Astrócitos/patologia , Proliferação de Células/efeitos dos fármacos , Conexina 43/antagonistas & inibidores , Agonistas de Aminoácidos Excitatórios/toxicidade , Genes src/fisiologia , Glucose/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Conexina 43/biossíntese , Conexina 43/genética , Ácido Caínico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
BACKGROUND: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation. RESULTS: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1. CONCLUSIONS: We mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.
Assuntos
Núcleo Celular/metabolismo , HIV-1/patogenicidade , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Sinais de Localização Nuclear/metabolismo , Vírus da Imunodeficiência Símia/patogenicidade , Síndrome da Imunodeficiência Adquirida/virologia , Transporte Ativo do Núcleo Celular , Alelos , Núcleo Celular/genética , Núcleo Celular/virologia , Citoplasma/metabolismo , Citoplasma/virologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Mutagênese Sítio-Dirigida , Mutação , Proteólise , Mapeamento por Restrição , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Células U937 , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.
Assuntos
Antivirais/isolamento & purificação , Técnicas Biossensoriais/métodos , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Células HEK293 , Humanos , Luciferases de Vaga-Lume/metabolismo , Mucosa Nasal/virologia , Pirazolonas/farmacologia , Piridonas/farmacologia , SARS-CoV-2/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.
Assuntos
Infecções por Arterivirus/tratamento farmacológico , Equartevirus/metabolismo , Ribavirina/farmacologia , Animais , Antivirais/farmacologia , Infecções por Arterivirus/veterinária , Linhagem Celular , Efeito Citopatogênico Viral/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Doenças dos Cavalos/virologia , Cavalos/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Purinas/antagonistas & inibidores , Purinas/biossíntese , Purinas/farmacologia , Pirimidinas/antagonistas & inibidores , Pirimidinas/biossíntese , Pirimidinas/farmacologia , RNA/farmacologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake.
Assuntos
Astrócitos/metabolismo , Proliferação de Células , Conexina 43/metabolismo , Endotelina-1/metabolismo , Junções Comunicantes/metabolismo , Glucose/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Comunicação Celular/fisiologia , Células Cultivadas , Conexina 43/genética , Ciclinas/metabolismo , Regulação para Baixo/fisiologia , Endotelina-1/farmacologia , Metabolismo Energético/fisiologia , Junções Comunicantes/efeitos dos fármacos , Transportador de Glucose Tipo 1/metabolismo , Hexoquinase/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Knockout , Fosforilação/efeitos dos fármacos , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Proteína do Retinoblastoma/metabolismoRESUMO
HIV persists in long-lived infected cells that are not affected by antiretroviral treatment. These HIV reservoirs are mainly located in CD4+ T cells, but their distribution is variable in the different subsets. Susceptibility to HIV-1 increases with CD4+ T cell differentiation. We evaluated whether the metabolic programming that supports the differentiation and function of CD4+ T cells affected their susceptibility to HIV-1. We found that differences in HIV-1 susceptibility between naive and more differentiated subsets were associated with the metabolic activity of the cells. Indeed, HIV-1 selectively infected CD4+ T cells with high oxidative phosphorylation and glycolysis, independent of their activation phenotype. Moreover, partial inhibition of glycolysis (1) impaired HIV-1 infection in vitro in all CD4+ T cell subsets, (2) decreased the viability of preinfected cells, and (3) precluded HIV-1 amplification in cells from HIV-infected individuals. Our results elucidate the link between cell metabolism and HIV-1 infection and identify a vulnerability in tackling HIV reservoirs.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/patologia , Diferenciação Celular , Células Cultivadas , Glicólise/imunologia , Infecções por HIV/patologia , HIV-1 , Humanos , Ativação Linfocitária , Fosforilação Oxidativa , Subpopulações de Linfócitos T/patologiaRESUMO
Spontaneous control of human immunodeficiency virus (HIV) is generally associated with an enhanced capacity of CD8+ T cells to eliminate infected CD4+ T cells, but the molecular characteristics of these highly functional CD8+ T cells are largely unknown. In the present study, using single-cell analysis, it was shown that HIV-specific, central memory CD8+ T cells from spontaneous HIV controllers (HICs) and antiretrovirally treated non-controllers have opposing transcriptomic profiles. Genes linked to effector functions and survival are upregulated in cells from HICs. In contrast, genes associated with activation, exhaustion and glycolysis are upregulated in cells from non-controllers. It was shown that HIV-specific CD8+ T cells from non-controllers are largely glucose dependent, whereas those from HICs have more diverse metabolic resources that enhance both their survival potential and their capacity to develop anti-HIV effector functions. The functional efficiency of the HIV-specific CD8+ T cell response in HICs is thus engraved in their memory population and related to their metabolic programme. Metabolic reprogramming in vitro through interleukin-15 treatment abrogated the glucose dependency and enhanced the antiviral potency of HIV-specific CD8+ T cells from non-controllers.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , HumanosRESUMO
SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition.