RESUMO
The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.
Assuntos
Moléculas de Adesão Celular , Vírus da Dengue , Evolução Molecular , Interações Hospedeiro-Patógeno , Receptores de Superfície Celular , Proteínas do Envelope Viral , Envelope Viral , Humanos , Brasil , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/química , Dengue/virologia , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Interações Hospedeiro-Patógeno/genética , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/química , Simulação de Dinâmica Molecular , Filogenia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/química , Receptores Virais/metabolismo , Receptores Virais/química , Receptores Virais/genética , Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/químicaRESUMO
Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.
Assuntos
Proteínas do Capsídeo , Vírus da Dengue , Humanos , Proteínas do Capsídeo/química , Vírus da Dengue/química , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Conformação Proteica em alfa-HéliceRESUMO
Dengue is a mosquito-borne disease endemic in many tropical and subtropical countries. It is caused by the dengue virus (DENV) that can be classified into 4 different serotypes (DENV-1-4). Early diagnosis and management can reduce morbidity and mortality rates of severe forms of the disease, as well as decrease the risk of larger outbreaks. Hiperendemicity in some regions of the world and the possibility that some people develop a more severe form of disease after a secondary infection caused by antibody-dependent enhancement justify the need to understand more thoroughly the antibody response induced against the virus. Here, we successfully produced a recombinant DENV-2 envelope (E) protein and its domains (EDI/II and EDIII) in two distinct expression systems: the Drosophila S2 insect cell system and the BL21 (DE3) pLySs bacterial system. We then evaluated the reactivity of sera from patients previously infected with DENV to each recombinant protein and to each domain separately. Our results show that the E protein produced in Drosophila S2 cells is recognized more frequently than the protein produced in bacteria. However, the recognition of E protein produced in bacteria correlates better with the DENV-2 sera neutralization capacity. The results described here emphasize the differences observed when antigens produced in bacteria or eukaryotic cells are used and may be useful to gain more insight into the humoral immune responses induced by dengue infection.
Assuntos
Vírus da Dengue , Dengue , Animais , Vírus da Dengue/metabolismo , Anticorpos Antivirais , Células Eucarióticas/metabolismo , Epitopos , Proteínas do Envelope Viral , Proteínas Recombinantes , Dengue/diagnóstico , Bactérias , Anticorpos NeutralizantesRESUMO
The dengue virus NS1 is a multifunctional protein that forms part of replication complexes. NS1 is also secreted, as a hexamer, to the extracellular milieu. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms. Cell binding and internalization of soluble NS1 result in endothelial hyperpermeability and in the downregulation of the innate immune response. In this work, we report that the HDL scavenger receptor B1 (SRB1) in human hepatic cells and a scavenger receptor B1-like in mosquito C6/36 cells act as cell surface binding receptors for dengue virus NS1. The presence of the SRB1 on the plasma membrane of C6/36 cells, as well as in Huh7 cells, was demonstrated by confocal microscopy. The internalization of NS1 can be efficiently blocked by anti-SRB1 antibodies, and previous incubation of the cells with HDL significantly reduces NS1 internalization. Significant reduction in NS1 internalization was observed in C6/36 cells transfected with siRNAs specific for SRB1. In addition, the transient expression of SRB1 in Vero cells, which lacks the receptor, allows NS1 internalization in these cells. Direct interaction between soluble NS1 and the SRB1 in Huh7 and C6/36 cells was demonstrated in situ by proximity ligation assays and in vitro by surface plasmon resonance. Finally, results are presented indicating that the SRB1 also acts as a cell receptor for Zika virus NS1. These results demonstrate that dengue virus NS1, a bona fide lipoprotein, usurps the HDL receptor for cell entry and offers explanations for the altered serum lipoprotein homeostasis observed in dengue patients. IMPORTANCE Dengue is the most common viral disease transmitted to humans by mosquitoes. The dengue virus NS1 is a multifunctional glycoprotein necessary for viral replication. NS1 is also secreted as a hexameric lipoprotein and circulates in high concentrations in the sera of patients. Circulating NS1 has been associated with dengue pathogenesis by several mechanisms, including favoring of virus replication in hepatocytes and dendritic cells and disruption of the endothelial glycocalyx leading to hyperpermeability. Those last actions require NS1 internalization. Here, we identify the scavenger cell receptor B1, as the cell-binding receptor for dengue and Zika virus NS1, in cultured liver and in mosquito cells. The results indicate that flavivirus NS1, a bona fide lipoprotein, usurps the human HDL receptor and may offer explanations for the alterations in serum lipoprotein homeostasis observed in dengue patients.
Assuntos
Vírus da Dengue , Receptores Depuradores , Proteínas não Estruturais Virais , Infecção por Zika virus , Zika virus , Animais , Linhagem Celular , Chlorocebus aethiops , Culicidae/virologia , Dengue/virologia , Vírus da Dengue/metabolismo , Humanos , Lipoproteínas HDL , Receptores de Lipoproteínas , Receptores Depuradores/metabolismo , Células Vero , Proteínas não Estruturais Virais/imunologia , Internalização do Vírus , Zika virus/metabolismoRESUMO
We synthesised and screened 18 aromatic derivatives of guanylhydrazones and oximes aromatic for their capacity to bind to dengue virus capsid protein (DENVC). The intended therapeutic target was the hydrophobic cleft of DENVC, which is a region responsible for its anchoring in lipid droplets in the infected cells. The inhibition of this process completely suppresses virus infectivity. Using NMR, we describe five compounds able to bind to the α1-α2 interface in the hydrophobic cleft. Saturation transfer difference experiments showed that the aromatic protons of the ligands are important for the interaction with DENVC. Fluorescence binding isotherms indicated that the selected compounds bind at micromolar affinities, possibly leading to binding-induced conformational changes. NMR-derived docking calculations of ligands showed that they position similarly in the hydrophobic cleft. Cytotoxicity experiments and calculations of in silico drug properties suggest that these compounds may be promising candidates in the search for antivirals targeting DENVC.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Vírus da Dengue/efeitos dos fármacos , Hidrazonas/farmacologia , Oximas/farmacologia , Antivirais/síntese química , Antivirais/química , Proteínas do Capsídeo/metabolismo , Vírus da Dengue/metabolismo , Relação Dose-Resposta a Droga , Hidrazonas/síntese química , Hidrazonas/química , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Oximas/síntese química , Oximas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The pathogenesis associated with Dengue virus (DENV) infection is marked by the impairment of host immune response. Consequently, the modulation of immune response has emerged as an important therapeutic target for the control of DENV infection. Vitamin D has been shown to regulate the immune response in DENV infection, although the molecular mechanism remains poorly understood. Post-transcriptional regulation of mRNA by miRNAs offers an opportunity to gain insight into the immunomodulation mediated by vitamin D. OBJECTIVE: Previously, it has been observed that a high dose of vitamin D (4000 IU) decreased DENV-2 infection and inflammatory response in monocyte-derived macrophages (MDMs). Here, we examine whether high or low doses of vitamin D supplements exert differential effect on miRNA expression in DENV-infected macrophages. METHODS: We analyzed miRNA expression profiles in MDMs isolated from healthy individuals who were given either 1000 or 4000 IU/day of vitamin D for 10 days. MDMs before or after vitamin D supplementation were challenged with DENV-2, and miRNAs profiles were analyzed by qPCR arrays. RESULTS: DENV-2 infected MDMs supplemented with 4000 IU, showed up-regulation of miR-374a-5p, miR-363-3p, miR-101-3p, miR-9-5p, miR-34a-5p, miR-200a-3p, and the family of miRNAs miR-21-5p, and miR-590-p. The miRNA profile and predicted target mRNAs suggested regulatory pathways in MDMs obtained from healthy donors who received higher doses of vitamin D. These DENV-2 infected MDMs expressed a unique set of miRNAs that target immune and cellular stress response genes. CONCLUSION: The results suggest vitamin D dose-dependent differential expression of miRNAs target key signaling pathways of the pathogenesis of dengue disease.
Assuntos
Vírus da Dengue , Dengue , MicroRNAs , Dengue/tratamento farmacológico , Dengue/genética , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Humanos , Macrófagos , MicroRNAs/genética , Replicação Viral , Vitamina D/metabolismo , Vitamina D/farmacologia , Vitamina D/uso terapêuticoRESUMO
The Dengue (DENV) and zika (ZIKV) virus infections are currently a public health concern. At present, there is no treatment or a safe and effective vaccine for these viruses. Hence, the development of new strategies as host-directed therapy is required. In this sense, Metformin (MET), an FDA-approved drug used for the treatment of type 2 diabetes, has shown an anti-DENV effect in vitro by activating AMPK and reducing HMGCR activity. In this study, MET treatment was evaluated during in vitro and in vivo ZIKV infection and compared to MET treatment during DENV infection. Our results demonstrated that MET has a broad in vitro antiviral spectrum. MET inhibited ZIKV infection in different cell lines, but it was most effective in inhibiting DENV and yellow fever virus (YFV) infection in Huh-7 cells. However, the drug failed to protect against ZIKV infection when AG129 immunodeficient mice were used as in vivo model. Interestingly, MET increased DENV-infected male mice's survival time, reducing the severe signs of the disease. Together, these findings indicate that, although MET was an effective antiviral agent to inhibit in vitro and in vivo DENV infection, it could only inhibit in vitro ZIKV infection.
Assuntos
Antivirais/uso terapêutico , Dengue/tratamento farmacológico , Metformina/uso terapêutico , Infecção por Zika virus/tratamento farmacológico , Animais , Antivirais/farmacologia , Linhagem Celular , Vírus da Dengue/isolamento & purificação , Vírus da Dengue/metabolismo , Reposicionamento de Medicamentos , Humanos , Metformina/farmacologia , Camundongos , Estudos Retrospectivos , Carga Viral , Proteínas Virais/biossíntese , Replicação Viral/efeitos dos fármacos , Zika virus/isolamento & purificação , Zika virus/metabolismoRESUMO
Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.
Assuntos
Apolipoproteína A-I/imunologia , Vírus da Dengue/metabolismo , Evasão da Resposta Imune/imunologia , Microdomínios da Membrana/metabolismo , Proteínas não Estruturais Virais/imunologia , Animais , Antivirais/farmacologia , Linhagem Celular , Colesterol/metabolismo , Dengue/patologia , Humanos , Inflamação/prevenção & controle , Camundongos , Peptídeos/farmacologia , Células RAW 264.7 , Ligação Viral/efeitos dos fármacosRESUMO
Dengue virus causes dengue hemorrhagic fever (DHF) and has been associated to fatal cases worldwide. The liver is one of the most important target tissues in severe cases, due to its intense viral replication and metabolic role. microRNAs role during infection is crucial to understand the regulatory mechanisms of DENV infection and can help in diagnostic and anti-viral therapies development. We sequenced the miRNome of six fatal cases and compared to five controls, to characterize the human microRNAs expression profile in the liver tissue during DHF. Eight microRNAs were differentially expressed, including miR-126-5p, a regulatory molecule of endothelial cells, miR-122-5p, a liver specific homeostasis regulator, and miR-146a-5p, an interferon-regulator. Enrichment analysis with predicted target genes of microRNAs revealed regulatory pathways of apoptosis, involving MAPK, RAS, CDK and FAS. Immune response pathways were related to NF- kB, CC and CX families, IL and TLR. This is the first description of the human microRNA and isomicroRNA profile in liver tissues from DHF cases. The results demonstrated the association of miR-126-5p, miR-122-5p and miR-146a-5p with DHF liver pathogenesis, involving endothelial repair and vascular permeability regulation, control of homeostasis and expression of inflammatory cytokines.
Assuntos
Vírus da Dengue/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Fígado/metabolismo , MicroRNAs/biossíntese , Dengue Grave/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Although dengue virus (DENV) replication occurs in the cytoplasm, the nucleus plays an essential role during infection. Both the capsid protein (C) and non-structural protein 5 (NS5) are translocated into the infected cell nucleus to favor viral replication. Previously, our group reported the nuclear localization of the NS3 protein during DENV infection of mosquito cells; however, the nuclear localization of the DENV NS3 protein in human host cells has not been described. Here, we demonstrated that NS3 is present in the nucleus of Huh7 cells at early infection times, and later, it is mainly located in the cytoplasm.