RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019, which has been a global pandemic. Since SARS-CoV-2 is transmitted through contaminated surfaces and aerosols, environmental disinfection is important to block the spread of the virus. Photocatalysts are attractive tools for virus inactivation and are widely used as air purifiers and coating materials. However, photocatalysts are inactive in the dark, and some of them need to be excited with light of a specific wavelength. Therefore, photocatalysts that can effectively inactivate SARS-CoV-2 in indoor environments are needed. Here, we show that a WO3 photocatalyst containing copper inactivated the SARS-CoV-2 WK-521 strain (Pango lineage A) upon irradiation with white light in a time- and concentration-dependent manner. Additionally, this photocatalyst also inactivated SARS-CoV-2 in dark conditions due to the antiviral effect of copper. Furthermore, this photocatalyst inactivated not only the WK-521 strain but also the Omicron variant BA.2. These results indicate that the WO3 photocatalyst containing copper can inactivate indoor SARS-CoV-2 regardless of the variant, in visible light or darkness, making it an effective tool for controlling the spread of SARS-CoV-2.
RESUMEN
Enterovirus 71 (EV71) is one of the causative agents of hand-foot-and-mouth disease, which in some circumstances could lead to severe neurological diseases. Despite of its importance for human health, little is known about the early stages of EV71 infection. EV71 starts uncoating with its receptor, human scavenger receptor B2 (hSCARB2), at low pH. We show that EV71 was not targeted to lysosomes in human rhabdomyosarcoma cells overexpressing hSCARB2 and that the autophagic pathway is not essential for EV71 productive uncoating. Instead, EV71 was efficiently uncoated 30â min after infection in late endosomes (LEs) containing hSCARB2, mannose-6-phosphate receptor (M6PR), RAB9, bis(monoacylglycero)phosphate and lysosomal associated membrane protein 2 (LAMP2). Furthering the notion that mature LEs are crucial for EV71 uncoating, cation-dependent (CD)-M6PR knockdown impairs EV71 infection. Since hSCARB2 interacts with cation-independent (CI)-M6PR through M6P-binding sites and CD-M6PR also harbor a M6P-binding site, CD-M6PR is likely to play important roles in EV71 uncoating in LEs.
Asunto(s)
Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Animales , Cationes/metabolismo , Endosomas/metabolismo , Enterovirus/metabolismo , Enterovirus Humano A/metabolismo , Humanos , Proteínas de Membrana de los Lisosomas/química , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismoRESUMEN
Inhibiting the programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) axis by monoclonal antibodies (mAbs) is a successful cancer immunotherapy. However, mAb-based drugs have various disadvantages including high production costs and large molecular sizes, which motivated us to develop a smaller alternative drug. Since PD-L1 binds PD-1 with moderate affinity, a higher affinity PD-1 variant should serve as a competitive inhibitor of the wild-type PD-1/PD-L1 interaction. In this report, we conducted in silico point mutagenesis of PD-1 to identify potent PD-1 variants with a higher affinity toward PD-L1 and refined the in silico results using a luciferase-based in-cell protein-protein interaction (PPI) assay. As a result, a PD-1 variant was developed that had two mutated amino acids (T76Y, A132V), termed 2-PD-1. 2-PD-1 could bind with PD-L1 at a dissociation constant of 12.74 nM. Moreover, 2-PD-1 successfully inhibited the PD-1/PD-L1 interaction with a half maximal inhibitory concentration of 19.15 nM and reactivated the T cell with a half maximal effective concentration of 136.1 nM. These results show that in silico mutagenesis combined with an in-cell PPI assay verification strategy successfully prepared a non-IgG inhibitor of the PD-1/PD-L1 interaction.
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Antígeno B7-H1/metabolismo , Proteínas de Punto de Control Inmunitario/farmacología , Receptor de Muerte Celular Programada 1/metabolismo , Unión Proteica/efectos de los fármacos , Simulación por Computador , Células HeLa , Humanos , Proteínas de Punto de Control Inmunitario/genética , Proteínas de Punto de Control Inmunitario/metabolismo , Activación de Linfocitos/efectos de los fármacos , Mutagénesis , Mutación Puntual , Receptor de Muerte Celular Programada 1/genética , Ingeniería de Proteínas , Linfocitos T/efectos de los fármacosRESUMEN
Current therapies for human immunodeficiency virus type 1 (HIV-1) do not completely eliminate viral reservoirs in cells, such as macrophages. The HIV-1 accessory protein viral protein R (Vpr) promotes virus production in macrophages, and the maintenance of Vpr is essential for HIV-1 replication in these reservoir cells. We identified two novel Vpr-binding proteins, i.e., protein arginine N-methyltransferases (PRMTs) 5 and 7, using human monocyte-derived macrophages (MDMs). Both proteins found to be important for prevention of Vpr degradation by the proteasome; in the context of PRMT5 and PRMT7 knockdowns, degradation of Vpr could be prevented using a proteasome inhibitor. In MDMs infected with a wild-type strain, knockdown of PRMT5/PRMT7 and low expression of PRMT5 resulted in inefficient virus production like Vpr-deficient strain infections. Thus, our findings suggest that PRMT5 and PRMT7 support HIV-1 replication via maintenance of Vpr protein stability.
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Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteína-Arginina N-Metiltransferasas/metabolismo , Replicación Viral , Secuencia de Aminoácidos , Proteínas Portadoras , Línea Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Macrófagos/virología , Unión Proteica , Proteína-Arginina N-Metiltransferasas/química , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.
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Colesterol/metabolismo , Proteínas Fúngicas/farmacología , Grifola/química , Microdominios de Membrana/efectos de los fármacos , Enfermedad de Niemann-Pick Tipo C/metabolismo , Esfingomielinas/metabolismo , Sitios de Unión , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Unión Proteica , Liberación del VirusRESUMEN
Nanostructured RNA carrying three different siRNAs was assembled to silence three target genes (Axin, APC, and GSK-3ß) in the Wnt/ß-catenin signaling pathway. The trimer RNA nanostructure included equimolar concentrations of three oligonucleotide sequences. The three armed structures and the size of the trimer RNA were confirmed by agarose gel electrophoresis, atomic force microscopy, and dynamic light scattering. In the presence of 10% human serum, the trimer RNA was able to resist degradation and maintained an intact structure for more than two hours. Protein expression analyses showed specific repression of the target proteins by siRNAs. As a result, the expression of luciferase in a ß-catenin reporter vector was significantly increased by the trimer RNA compared with a pool of the three individual siRNAs. This high activity at a low concentration was considered to be due to the 3-in-1 format of the trimer and the long-term resistance to serum proteins by nanostructure formation. We demonstrated that a nanostructured "3-in-1" siRNA is effective in enhancing the effect of RNA interference.
RESUMEN
High aspect ratio nanomaterials, such as vertically aligned silicon nanowire (SiNW) substrates, are three-dimensional topological features for cell manipulations. A high density of SiNWs significantly affects not only cell adhesion and proliferation but also the delivery of biomolecules to cells. Here, we used polydopamine (PD) that simply formed a thin coating on various material surfaces by the action of dopamine as a bioinspired approach. The PD coating not only enhanced cell adhesion, spreading, and growth but also anchored more siRNA by adsorption and provided more surface concentration for substrate-mediated delivery. By comparing plain and SiNW surfaces with the same amount of loaded siRNA, we quantitatively found that PD coating efficiently anchored siRNA on the surface, which knocked down the expression of a specific gene by RNA interference. It was also found that the interaction of SiNWs with the cell membrane perturbed the lateral diffusion of lipids in the membrane by fluorescence recovery after photobleaching. The perturbation was considered to induce the effective delivery of siRNA into cells and allow the cells to carry out their biological functions. These results suggest promising applications of PD-coated, high-density SiNWs as simple, fast, and versatile platforms for transmembrane delivery of biomolecules.
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Nanocables , Membrana Celular , Silenciador del Gen , Indoles , Polímeros , ARN Interferente PequeñoRESUMEN
Viral protein R (Vpr) is an accessory gene product of human immunodeficiency virus type 1 (HIV-1) that plays multiple important roles associated with viral replication. Structural studies using NMR have revealed that Vpr consists of three α-helices and contains flexible N- and C-termini. However, the molecular mechanisms associated with Vpr function have not been elucidated. To investigate Vpr multifunctionality, we performed an X-ray crystallographic study of Vpr complexes containing importin-α, a known Vpr binding partner present in host cells. Elucidation of the crystal structure revealed that the flexible C-terminus changes its conformation to a twisted ß-turn via an induced-fit mechanism, enabling binding to a minor nuclear localization signal (NLS) site of importin-α. The Vpr C-terminus can also bind with major NLS sites of importin-α in an extended conformation in different ways. These results, which represent the first reported crystallographic analysis of Vpr, demonstrate the multifunctional aspects that enable Vpr interaction with a variety of cellular proteins.
Asunto(s)
Productos del Gen vpr/metabolismo , VIH-1/metabolismo , Unión Proteica/fisiología , alfa Carioferinas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Humanos , Señales de Localización Nuclear/metabolismo , Replicación Viral/fisiologíaRESUMEN
The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54-74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Hematoxilina/química , Macrófagos/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Fármacos Anti-VIH/farmacología , Células Cultivadas , Infecciones por VIH/virología , Humanos , Macrófagos/virologíaRESUMEN
Owing to its hydrophilicity, negative charge, small size, and labile degradation by endogenous nucleases, small interfering RNA (siRNA) delivery must be achieved by a carrier system. In this study, cationic copolymers composed of low-molecular-weight polyethylenimine and polythiophenes were synthesized and evaluated as novel self-tracking siRNA delivery vectors. The concept underlying the design of these copolymers is that hydrophobicity and rigidity of polythiophenes should enhance the transport of siRNA across the cell membrane and endosomal membrane. A gel retardation assay showed that the nanosized complexes formed between the copolymers and siRNA were stable even at a molar ratio of 1 : 2. The high cellular uptake (>80%) and localization of the copolymer vectors inside the cells were easily analyzed by tracking the fluorescence of polythiophene using fluorescent microscopy and cytometry. An in vitro luciferase knockdown (KD) assay in A549-luc cells demonstrated that the siRNA complexes with more hydrophobic copolymers achieved a higher KD efficiency of 52.8% without notable cytotoxicity, indicating protein-specific KD activity rather than solely the cytotoxicity of the materials. Our polythiophene copolymers should serve as novel, efficient, low cell toxicity, and label-free siRNA delivery systems.
Asunto(s)
Técnicas de Transferencia de Gen , Polietileneimina/administración & dosificación , Polímeros/administración & dosificación , ARN Interferente Pequeño/genética , Tiofenos/administración & dosificación , Cationes/administración & dosificación , Cationes/química , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Vectores Genéticos , Humanos , Peso Molecular , Polietileneimina/química , Polímeros/química , ARN Interferente Pequeño/administración & dosificación , Tiofenos/química , TransfecciónRESUMEN
Numerous cationic peptides that penetrate cells have been studied intensively as drug delivery system carriers for cellular delivery. However, cationic molecules tend to be cytotoxic and cause inflammation, and their stability in the blood is usually low. We have previously demonstrated that a rigid and fibrous cationic coiled-coil protein exhibited cell-penetrating ability superior to that of previously reported cell-penetrating peptides. Making use of structural properties, here we describe the cell-penetrating activity of a rigid and fibrous coiled-coil protein with a noncationic surface. A fibrous coiled-coil protein of pI 6.5 penetrated 100% of the cells tested in vitro at a concentration of 500 nM, which is comparable to that of previously reported cell-penetrating peptides. We also investigated the effect of cell-strain dependency and short-term cytotoxicity.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Secuencia de Aminoácidos , Anisotropía , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Células K562 , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
Molecules with structural anisotropy and rigidity, such as asbestos, demonstrate high cell-penetrating activity but also high toxicity. Here we synthesize a biodegradable, rigid, and fibrous artificial protein, CCPC 140, as a potential vehicle for cellular delivery. CCPC 140 penetrated 100% of cells tested in vitro, even at a concentration of 3.1 nM-superior to previously reported cell-penetrating peptides. The effects of cell-strain-dependency and aspect ratio on the cell-penetrating activity of CCPC 140 were also investigated.
Asunto(s)
Péptidos de Penetración Celular/química , Secuencia de Aminoácidos , Animales , Anisotropía , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos/síntesis química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Células HeLa , Humanos , Masculino , Fenómenos Mecánicos , Datos de Secuencia Molecular , Conejos , Tropomiosina/químicaRESUMEN
The nanoparticle-based delivery of siRNA with a noncationic outermost surface at a low particle concentration is greatly desired. We newly synthesized a bifurcated ligand (BL) possessing hydrophobic and hydrophilic arms as a surface ligand for gold nanoparticles (AuNPs) to allow siRNA delivery. The concept underlying the design of this ligand is that amphiphilic property should allow AuNPs to permeate the cell cytosol thorough the endosomal membrane. BLs and quaternary cationic ligands were codisplayed on 40 nm AuNPs, which were subsequently coated with siRNA via electrostatic interaction. The number of siRNAs immobilized on a single nanoparticle was 26, and the conjugate showed a negative zeta potential due to siRNAs on the outermost surface of the AuNPs. Apparent gene silencing of luciferase expression in HeLa cells was achieved at an AuNP concentration as low as 60 pM. Almost no gene silencing was observed for AuNPs not displaying BLs. To reveal the effect of the BL, we compared the number of AuNPs internalized into HeLa cells and the localization in the cytosol between AuNPs displaying and those not displaying BLs. These analyses indicated that the role of BLs is not only the simple promotion of cellular uptake but also involves the enhancement of AuNPs permeation into the cytosol from the endosomes, leading to effective gene silencing.
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Endosomas/química , Oro/química , Nanopartículas del Metal/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Transfección/métodos , Difusión , Silenciador del Gen , Células HeLa , Humanos , Ligandos , Nanopartículas del Metal/ultraestructura , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Nanocápsulas/ultraestructura , Tamaño de la Partícula , ARN Interferente Pequeño/administración & dosificación , Tensoactivos/químicaRESUMEN
Gold nanoparticles (AuNPs) coated with ester-headed or ether-headed PEG ligands were synthesized. Ester-headed AuNPs, but not ether-headed, were transferred from the organic phase (CH2Cl2) to the alkali aqueous phase, indicating that the hydrolysis of the ester moiety triggered the phase transfer of the AuNPs. We found that AuNPs with ester-headed ligands (ester-AuNPs) were internalized into HeLa cells at a greater level than were ether-headed AuNPs.
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Ésteres/química , Oro/química , Nanopartículas del Metal/química , Células HeLa , Humanos , Espectrofotometría AtómicaRESUMEN
Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcription and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phosphorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine putative phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant. Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron microscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphorylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these results indicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation.
Asunto(s)
Virus del Sarampión/metabolismo , Sarampión/virología , Nucleocápside/metabolismo , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Treonina/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Humanos , Virus del Sarampión/química , Virus del Sarampión/genética , Datos de Secuencia Molecular , Nucleocápside/genética , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Fosforilación , Treonina/genética , Proteínas Virales/genéticaRESUMEN
The nucleoprotein (NP) of influenza A virus is transported into the nucleus via the classical importin α/ß pathway, and proceeds via nuclear localization signals (NLSs) recognized by importin α molecules. Although NP binds to importin α isoforms Rch1, Qip1 and NPI-1, the role of each individual isoform during the nuclear transport of NP and replication of the influenza virus remains unknown. In this study, we examined the contribution of importin α isoforms for nuclear localization of NP and viral growth using a panel of NP mutants containing serial alanine replacements within an unconventional NLS of NP. Alanine mutation at amino acid 8 (R8A) caused a significant reduction in the nuclear localization and binding to the three importin isoforms. The R8A NP mutant virus did not generate by reverse-genetics approach. This indicates that position 8 is the main site that mediates nuclear localization via interactions with Rch1, Qip1 and NPI-1, and subsequent viral production. This was confirmed by the finding that the conservation of amino acid 8 in human- and avian-origin influenza virus NP was necessary for virus propagation. By contrast, another mutant, S9A NP, which localized in the nucleus, caused a reduction in viral growth and vRNA transcription, suggesting that the unconventional NLS within NP may be associated with nuclear transport, vRNA transcription and viral replication through independent pathways. Interestingly, the N-terminal 110-amino acid region, which contained the unconventional NLS with S9A mutation, mainly bound to Qip1. Furthermore, activities of vRNA transcription and replication of S9A NP mutants were decreased by silencing Qip1 in without changing nuclear localization, indicating that Qip1 involves in multiplication of S9A mutant virus independently of nuclear transport function. Collectively, our results demonstrate the unconventional NLS within NP might have the additional ability to regulate the viral replication that is independent of nuclear localization activity via interactions with Qip1.
Asunto(s)
Transporte Activo de Núcleo Celular , Alanina/genética , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Mutación , alfa Carioferinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Codón , Silenciador del Gen , Humanos , Gripe Humana/metabolismo , Gripe Humana/virología , Señales de Localización Nuclear/química , Señales de Localización Nuclear/genética , Proteínas de la Nucleocápside , Unión Proteica , Transporte de Proteínas , ARN Viral , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Replicación Viral/genéticaRESUMEN
Influenza viruses have developed resistance to current drugs, creating a need for new antiviral targets and new drugs to treat influenza virus infections. In this study, computational and experimental screening of an extensive compound library identified THC19, which was able to suppress influenza virus replication. This compound had no cytotoxic effects and did not disrupt cell cycle progression or induce apoptosis in MDCK cells as confirmed by WST-1 assays, flow cytometry analysis, and caspase-3 assays. Time-of-addition experiments showed that THC19 acts at a relatively early stage of the viral lifecycle. Subsequent mini-genome assays revealed that THC19 inhibited viral genome replication and/or transcription, suggesting that it interferes with one or more of the viral components that form the ribonucleoprotein complexes, namely polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP) and viral RNA. Finally, mini-genome assays where PB2, PB1, PA or NP from A/WSN/33 (H1N1) virus were replaced with those from A/Udorn/307/1972 (H3N2) virus effectively demonstrated that THC19 inhibited viral multiplication in a manner dependent upon the PA subunit. Taken together, these results suggest that influenza virus PA protein is a potential target for, and may aid the development of, novel compounds that inhibit influenza A virus replication.
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Antivirales/aislamiento & purificación , Antivirales/farmacología , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/efectos de los fármacos , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Animales , Antivirales/efectos adversos , Línea Celular , Perros , Inhibidores Enzimáticos/efectos adversos , Humanos , Pruebas de Sensibilidad Microbiana , Transcripción Genética/efectos de los fármacosRESUMEN
SAR studies for the exploration a novel class of anti-human immunodeficiency virus type 1 (HIV-1) agents based on the hematoxylin structure (1) are described. The systematic deoxygenations of 1 including asymmetric synthesis were conducted to obtain a compound showing high potencies for inhibiting the nuclear import and viral replication as anti-HIV-1 agent. Among all, C-3-deoxygenated analog 16 exhibited most promising biological activities as anti-HIV-1 agent such as lower cytotoxicity (16:1; >80:40 µM), stronger inhibition of nuclear import (0.5:1.3 µM), and viral replication in HIV-1-infected TZM-bl cells (24.6:100 µM), human peripheral blood mononuclear cells (PMBCs) (30.1 µM: toxic). Different spectra of inhibitory activities against infected three healthy humans macrophages with high (donor A) and low (donor B and C) amounts of virus were also observed. Thus 16 showed 10-times stronger activity than 1 (16:1; 0.1:<1.0 µM) in the case of A, while 16 and 1 showed comparable activities in the cases of B and C (>0.01 and >0.00 1µM). The comparison of the inhibition of viral p24 antigen production was clearly indicated that compound 16 is at least twofold more potent anti-viral activity than 1. Thus, structures and actions of deoxy analogs particularly 16 could provide valuable information for the development of a novel class of anti-HIV-1 agents.
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Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Hematoxilina/síntesis química , Fármacos Anti-VIH/química , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Hematoxilina/química , Hematoxilina/farmacología , Humanos , Estructura Molecular , Oxígeno/químicaRESUMEN
Many viruses use their host's cellular machinery to regulate the functions of viral proteins. The phosphorylation of viral proteins is known to play a role in genome transcription and replication in paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected cells, is a component of the N-RNA complex and supports the transcription and replication of virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in steady-state cells, but was detected after inhibition of cellular protein phosphatases. We identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by electrospray ionization-quadrupole time-of-flight mass spectrometry. In the NiV minigenome assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted in a reduction in luciferase activity of approximately 45â% compared with the wild-type protein. Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a more significant decrease in luciferase activity - approximately 81â%. Northern blot analysis showed that both virus transcription and replication were reduced by these mutations. These results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in virus transcription and replication.
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Virus Nipah/fisiología , Nucleoproteínas/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Replicación Viral , Animales , Línea Celular , Chlorocebus aethiops , Humanos , Fosforilación , ARN Viral/metabolismo , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.