RESUMO
Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents the largest current health challenge for the society. At the moment, the therapeutic strategies to deal with this disease are only supportive. It is well known that zinc (Zn) possesses a variety of direct and indirect antiviral properties, which are realized through different mechanisms. Administration of Zn supplement has a potential to enhance antiviral immunity, both innate and humoral, and to restore depleted immune cell function or to improve normal immune cell function, in particular in immunocompromised or elderly patients. Zn may also act in a synergistic manner when co-administered with the standard antiviral therapy, as was demonstrated in patients with hepatitis C, HIV, and SARS-CoV-1. Effectiveness of Zn against a number of viral species is mainly realized through the physical processes, such as virus attachment, infection, and uncoating. Zn may also protect or stabilize the cell membrane which could contribute to blocking of the virus entry into the cell. On the other hand, it was demonstrated that Zn may inhibit viral replication by alteration of the proteolytic processing of replicase polyproteins and RNA-dependent RNA polymerase (RdRp) in rhinoviruses, HCV, and influenza virus, and diminish the RNA-synthesizing activity of nidoviruses, for which SARS-CoV-2 belongs. Therefore, it may be hypothesized that Zn supplementation may be of potential benefit for prophylaxis and treatment of COVID-19.
Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/prevenção & controle , Suplementos Nutricionais , Zinco/uso terapêutico , Antivirais/uso terapêutico , Membrana Celular/virologia , Humanos , Imunidade Humoral , Imunidade Inata , Modelos Teóricos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Resultado do Tratamento , Replicação ViralRESUMO
Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.
Assuntos
Antivirais/farmacologia , Membrana Celular/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Expressão Gênica/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/virologia , Masculino , Medicina Tradicional Chinesa/métodos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/virologiaRESUMO
Astaxanthin (Asta), red pigment of the carotenoid family, is known for its anti-oxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties. In this study, we evaluated the effects of Asta on isolated human sperm in the presence of human papillomavirus (HPV) 16 capsid protein, L1. Sperm, purified by gradient separation, were treated with HPV16-L1 in both a dose and time-dependent manner in the absence or presence of 30 min-Asta pre-incubation. Effects of HPV16-L1 alone after Asta pre-incubation were evaluated by rafts (CTB) and Lyn dislocation, Tyr-phosphorylation (Tyr-P) of the head, percentages of acrosome-reacted cells (ARC) and endogenous reactive oxygen species (ROS) generation. Sperm membranes were also analyzed for the HPV16-L1 content. Results show that HPV16-L1 drastically reduced membrane rearrangement with percentage of sperm showing head CTB and Lyn displacement decreasing from 72% to 15.8%, and from 63.1% to 13.9%, respectively. Accordingly, both Tyr-P of the head and ARC decreased from 68.4% to 10.2%, and from 65.7% to 14.6%, respectively. Asta pre-incubation prevented this drop and restored values of the percentage of ARC up to 40.8%. No alteration was found in either the ROS generation curve or sperm motility. In conclusion, Asta is able to preserve sperm by reducing the amount of HPV16-L1 bound onto membranes.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Papillomavirus Humano 16/patogenicidade , Proteínas Oncogênicas Virais/metabolismo , Espermatozoides/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/virologia , Clorofíceas/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/virologia , Xantofilas/farmacologia , Xantofilas/uso terapêuticoRESUMO
Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Impedância Elétrica , Fusão de Membrana/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Células COS , Fusão Celular , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/virologia , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Vírus da Dengue/ultraestrutura , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Fusão de Membrana/fisiologia , Microscopia Eletrônica de Varredura , Piridinas/farmacologia , Vírus Sinciciais Respiratórios/fisiologia , Vírus Sinciciais Respiratórios/ultraestrutura , Células VeroRESUMO
Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Fusão de HIV/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Virologia/métodos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , HIV-1/fisiologia , Humanos , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
HIV-Tat has been demonstrated to be secreted from cells in a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent manner. Here we show that HIV-Tat forms membrane-inserted oligomers, a process that is accompanied by changes in secondary structure with a strong increase in antiparallel ß sheet content. Intriguingly, oligomerization of HIV-Tat on membrane surfaces leads to the formation of membrane pores, as demonstrated by physical membrane passage of small fluorescent tracer molecules. Although membrane binding of HIV-Tat did not strictly depend on PI(4,5)P2 but, rather, was mediated by a range of acidic membrane lipids, a functional interaction between PI(4,5)P2 and HIV-Tat was critically required for efficient membrane pore formation by HIV-Tat oligomers. These properties are strikingly similar to what has been reported previously for fibroblast growth factor 2 (FGF2), providing strong evidence of a common core mechanism of unconventional secretion shared by HIV-Tat and fibroblast growth factor 2.
Assuntos
Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Membrana Celular/virologia , Eletroforese em Gel de Poliacrilamida , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Espectroscopia de Infravermelho com Transformada de Fourier , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
The formation of plasma membrane (PM) microdomains plays a crucial role in the regulation of membrane signaling and trafficking. Remorins are a plant-specific family of proteins organized in six phylogenetic groups, and Remorins of group 1 are among the few plant proteins known to specifically associate with membrane rafts. As such, they are valuable to understand the molecular bases for PM lateral organization in plants. However, little is known about the structural determinants underlying the specific association of group 1 Remorins with membrane rafts. We used a structure-function approach to identify a short C-terminal anchor (RemCA) indispensable and sufficient for tight direct binding of potato (Solanum tuberosum) REMORIN 1.3 (StREM1.3) to the PM. RemCA switches from unordered to α-helical structure in a nonpolar environment. Protein structure modeling indicates that RemCA folds into a tight hairpin of amphipathic helices. Consistently, mutations reducing RemCA amphipathy abolished StREM1.3 PM localization. Furthermore, RemCA directly binds to biological membranes in vitro, shows higher affinity for Detergent-Insoluble Membranes lipids, and targets yellow fluorescent protein to Detergent-Insoluble Membranes in vivo. Mutations in RemCA resulting in cytoplasmic StREM1.3 localization abolish StREM1.3 function in restricting potato virus X movement. The mechanisms described here provide new insights on the control and function of lateral segregation of plant PM.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Potexvirus/metabolismo , Solanum tuberosum/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Proteínas de Transporte/genética , Membrana Celular/genética , Membrana Celular/virologia , Dicroísmo Circular , Clonagem Molecular , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Fosfoproteínas/genética , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Proteínas de Plantas/genética , Potexvirus/patogenicidade , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Transporte Proteico , Solanum tuberosum/genética , Solanum tuberosum/virologia , Relação Estrutura-AtividadeRESUMO
The biosynthetic pathway carries cargos from the endoplasmic reticulum (ER) to the trans Golgi network (TGN) via a typical passage through the Golgi. Interestingly, large particles such as procollagen, chylomicrons and some viruses all reach the TGN by atypical routes. Given this dichotomy, we anticipated that such cargos might rely on non-classical machineries downstream of the TGN. Using Herpes simplex virus type 1 (HSV-1) as a model and a synchronized infection protocol that focuses on TGN to plasma membrane transport, the present study revealed the surprising implication of the cellular serine-threonine protein kinase D in HSV-1 egress. These findings, confirmed by a variety of complementary means [pharmacological inhibitors, dominant negative mutant, RNA interference and electron microscopy (EM)], identify one of possibly several cellular factors that modulate the egress of viruses transiting at the TGN. Moreover, the involvement of this kinase, previously known to regulate the transport of small basolateral cargos, highlights the trafficking of both small and exceptionally large entities by a common machinery downstream of the TGN, in sharp contrast to earlier steps of transport. Conceptually, this indicates the TGN is not only a sorting station from which cargos can depart towards different destinations but also a meeting point where conventional and unconventional routes can meet along the biosynthetic pathway. Lastly, given the apical release of HSV-1 in neurons, it opens up the possibility that this kinase might regulate some apical sorting.
Assuntos
Capsídeo/metabolismo , Membrana Celular , Herpesvirus Humano 1/metabolismo , Proteína Quinase C/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Diglicerídeos/antagonistas & inibidores , Diglicerídeos/química , Diglicerídeos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Herpes Simples/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Rede trans-Golgi/ultraestruturaRESUMO
Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.
Assuntos
Membrana Celular/virologia , HIV-1/metabolismo , Proteínas Virais/química , Tirosina Quinase da Agamaglobulinemia , Apoptose , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Sobrevivência Celular , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Protein transduction therapy is a newly developing method that allows proteins, peptides, and biologically active compounds to penetrate across the plasma membrane by being fused with cell-penetrating peptides such as polyarginine. Polyarginine-fused p53 protein penetrates across the plasma membrane of cancer cells and inhibits the growth of the cells. However, the protein is often entrapped inside macropinosomes in the cytoplasm. Therefore, high dose concentrations of the protein are needed for it to function effectively. To overcome this problem, in the present study, polyarginine-fused p53 was linked with the NH(2)-terminal domain of influenza virus hemagglutinin-2 subunit (HA2), which is a pH-dependent fusogenic peptide that induces the lysis of membranes at low pH levels. The protein was capable of efficiently translocating into the nucleus of glioma cells and induced p21(WAF1) transcriptional activity more effectively than did polyarginine-fused p53 protein. Moreover, low concentrations of the protein significantly inhibited the growth of cancer cells. These results suggest that protein transduction therapy using polyarginine and HA2 may be useful as a method for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Astrócitos/metabolismo , Sinergismo Farmacológico , Hemaglutininas Virais/química , Neoplasias/tratamento farmacológico , Orthomyxoviridae/metabolismo , Peptídeos/química , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Astrócitos/efeitos dos fármacos , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Membrana Celular/virologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Fluoresceína-5-Isotiocianato/farmacologia , Genes Reporter , Humanos , Concentração de Íons de Hidrogênio , Marcação In Situ das Extremidades Cortadas , Mutagênese Sítio-Dirigida , Neoplasias/virologia , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/química , Fatores de Tempo , Ativação Transcricional , Proteína Supressora de Tumor p53/químicaRESUMO
Rubella virus (RV) envelope glycoproteins E1 and E2 are targeted to the Golgi as heterodimers. While E2 contains a transmembrane Golgi retention signal, E1 is arrested in a pre-Golgi compartment in the absence of E2, and appears to require heterodimerization in order to reach the Golgi. Various forms of E1 with deletions in the ectodomain or lacking the cytoplasmic (CT) and transmembrane (TM) domains, as well as the 29 C-terminal amino acid residues of the ectodomain were also retained intracellularly. We therefore investigated the possibility of targetting E1 to the plasma membrane by addition of a glycosylphosphatidylinositol (GPI) anchor. We found that E1GPI was transported to the cell surface where it retained the hemadsorption activity characteristic of the wild-type E1/E2 heterodimer. Furthermore, coexpression of a mammalian GPI-specific phospholipase D (GPI-PLD) resulted in the release of E1GPI and in constitutive expression of a soluble form of E1. This study thus demonstrates that the GPI anchor has a dominant effect over the E1 pre-Golgi retention signal and that E1 is sufficient for hemadsorption.